Compositions and methods for 3-hydroxypropionic acid production

ABSTRACT

The present application discloses genetically modified yeast cells comprising an active 3-HP fermentation pathway, and the use of these cells to produce 3-HP.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/301,556, filed Nov. 21, 2011, which claims priority benefit of U.S.Provisional Application No. 61/416,199, filed Nov. 22, 2010, and U.S.Provisional Application No. 61/535,181, filed Sep. 15, 2011. The contentof these applications is hereby incorporated by reference as if it wasset forth in full below.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND

3-hydroxypropionic acid (3-HP) is a three carbon carboxylic acididentified by the U.S. Department of Energy as one of the top 12high-potential building block chemicals that can be made byfermentation. Alternative names for 3-HP, which is an isomer of lactic(2-hydroxypropionic) acid, include ethylene lactic acid and3-hydroxypropionate. 3-HP is an attractive renewable platform chemical,with 100% theoretical yield from glucose, multiple functional groupsthat allow it to participate in a variety of chemical reactions, and lowtoxicity. 3-HP can be used as a substrate to form several commoditychemicals, such as 1,3-propanediol, malonic acid, acrylamide, andacrylic acid. Acrylic acid is a large-volume chemical (>7 billionlbs/year) used to make acrylate esters and superabsorbent polymers, andis currently derived from catalytic oxidation of propylene. Fermentativeproduction of 3-HP would provide a sustainable alternative topetrochemicals as the feedstock for these commercially-significantchemicals, thus reducing energy consumption, US dependence on foreignoil, and the production of greenhouse gases.

Bacteria can be used to ferment sugars to organic acids. However,bacteria present certain drawbacks for large-scale organic acidproduction. As organic acids are produced, the fermentation mediumbecomes increasingly acidic. Lower pH conditions are actuallypreferable, because the resultant product is partially or wholly in theacid form. However, most bacteria that produce organic acids do notperform well in strongly acidic environments, and therefore either dieor begin producing so slowly that they become economically unviable asthe medium becomes more acidic. To prevent this, it becomes necessary tobuffer the medium to maintain a higher pH. However, this makes recoveryof the organic acid product more difficult and expensive.

There has been increasing interest in recent years around the use ofyeast to ferment sugars to organic acids. Yeasts are used asbiocatalysts in a number of industrial fermentations, and presentseveral advantages over bacteria. While many bacteria are unable tosynthesize certain amino acids or proteins that they need to grow andmetabolize sugars efficiently, most yeast species can synthesize theirnecessary amino acids or proteins from inorganic nitrogen compounds.Yeasts are also not susceptible to bacteriophage infection, which canlead to loss of productivity or of whole fermentation runs in bacteria.

Although yeasts are attractive candidates for organic acid production,they present several difficulties. First, pathway engineering in yeastis typically more difficult than in bacteria. Enzymes in yeast arecompartmentalized in the cytoplasm, mitochondria, or peroxisomes,whereas in bacteria they are pooled in the cytoplasm. This means thattargeting signals may need to be removed to ensure that all the enzymesof the biosynthetic pathway co-exist in the same compartment within asingle cell. Control of transport of pathway intermediates between thecompartments may also be necessary to maximize carbon flow to thedesired product. Second, not all yeast species meet the necessarycriteria for economic fermentation on a large scale. In fact, only asmall percentage of yeasts possess the combination of sufficiently highvolumetric and specific sugar utilization with the ability to growrobustly under low pH conditions. The U.S. Department of Energy hasestimated that production rates of approximately 2.5 g/L/hour arenecessary for economic fermentations of several organic acids, including3-HP (http://www1.eere.energy.gov/biomass/pdfs/35523.pdf).

Although many yeast species naturally ferment hexose sugars to ethanol,few if any naturally produce significant yields of organic acids. Thishas led to efforts to genetically modify various yeast species toproduce organic acids. Genetically modified yeast strains that producelactic acid have been previously developed by disrupting or deleting thenative pyruvate decarboxylase (PDC) gene and inserting a lactatedehydrogenase (LDH) gene to eliminate ethanol production (see, e.g.,WO99/14335, WO00/71738, WO02/42471, WO03/049525, WO03/102152 andWO03/102201). This alteration diverts sugar metabolism from ethanolproduction to lactic acid production. The fermentation products andpathways for yeast differ from those of bacteria, and thus differentengineering approaches are necessary to maximize yield. Other nativeproducts that may require elimination or reduction in order to enhanceorganic acid product yield or purity are glycerol, acetate, and diols.The reduction of glycerol in genetically altered yeast strains isdescribed in, for example, WO07/106524.

Unlike lactic acid, 3-HP is not a major end product of any pathway knownin nature, being found in only trace amounts in some bacteria and fungi.Thus, a greater deal of genetic engineering is necessary to generateyeast that produce 3-HP. A Saccharomyces cerevisiae strain waspreviously engineered to produce 3-HP at very low levels through alactate intermediate (see WO02/042418). However, the tolerance level ofwild-type S. cerevisiae is insufficient to make it an optimal host for3-HP production. Therefore, there is a need for improved yeast strainsthat generate 3-HP in a more cost-effective manner on an industrialscale.

SUMMARY

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from PEP, pyruvate,and/or glycerol to 3-HP. In certain embodiments, the cells providedherein contain one or more 3-HP pathway genes encoding enzymes with PPC,PYC, AAT, ADC, BAAT, gabT, 3-HPDH, HIBADH, 4-hydroxybutyratedehydrogenase, ACC, AAM, alanine dehydrogenase, aldehyde dehydrogenase,BCKA, KGD, 4-aminobutyrate aminotransferase, β-alanyl-CoA ammonia lyase,Co-A acylating malonate semialdehyde dehydrogenase, CoA synthetase, CoAtransferase, glycerol dehydratase, IPDA, LDH, lactyl-CoA dehydratase,malate decarboxylase, malate dehydrogenase, malonyl-CoA reductase, OAAformatelyase, OAA dehydrogenase, pyruvate/alanine aminotransferase, PDH,2-keto acid decarboxylase, 3-HP-CoA dehydratase, 3-HP-CoA hydrolase, or3-hydroxyisobutyryl-CoA hydrolase activity.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from PEP orpyruvate to 3-HP, wherein the cells contain one or more genes encodingenzymes with PPC, PYC, AAT, ADC, BAAT, gabT, 3-HPDH, HIBADH, and4-hydroxybutyrate dehydrogenase activity. In certain embodiments, one ormore of the 3-HP pathway genes are exogenous, and in these embodimentsthe genes may be derived from a yeast, fungal, bacterial, plant, insect,or mammalian source. For example, the cells may contain a yeast PYC genederived from I. orientalis or a bacterial PYC gene derived from R.sphaeroides, R. etli, P. fluorescens, C. glutamicum, or S. meliloti; abacterial PPC gene derived from E. coli, M. thermoautotrophicum, or C.perfringens, a yeast AAT gene derived from I. orientalis or S.cerevisiae or a bacterial AAT gene derived from E. coli; a bacterial ADCgene derived from S. avermitilis, C. acetobutylicum, H. pylori, B.licheniformis, or C. glutamicum; a yeast BAAT gene derived from I.orientalis or S. kluyvenri or a bacterial BAAT gene derived from S.avermitilis; a yeast gabT gene derived from S. cerevisiae or a bacterialgabT gene derived from S. avermitilis; a yeast 3-HPDH gene derived fromI. orientalis or S. cerevisiae or a bacterial 3-HPDH gene derived fromE. coli or M. sedula; a bacterial HIBADH gene derived from A. faecalis,P. putida, or P. aeruginosa; and/or a yeast 4-hydroxybutyratedehydrogenase gene derived from C. kluyveri or a bacterial4-hydroxybutyrate dehydrogenase gene derived from R. eutropha.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from PEP orpyruvate to 3-HP, wherein the cells contain one or more genes encodingenzymes with PPC, malate dehydrogenase, and malate decarboxylaseactivity. In certain embodiments, one or more of the 3-HP pathway genesare exogenous, and in these embodiments the genes may be derived from ayeast, fungal, bacterial, plant, insect, or mammalian source.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from PEP orpyruvate to 3-HP, wherein the cells contain one or more genes encodingenzymes with PPC, 2-keto acid decarboxylase, KGD, BCKA, indolepyruvatedecarboxylase, 3-HPDH, HIBADH, or 4-hydroxybutyrate dehydrogenaseactivity. In certain embodiments, one or more of the 3-HP pathway genesare exogenous, and in these embodiments the genes may be derived from ayeast, fungal, bacterial, plant, insect, or mammalian source.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from PEP orpyruvate to 3-HP, wherein the cells contain one or more genes encodingenzymes with PPC, OAA formatelyase, malonyl-CoA reductase, Co-Aacylating malonate semialdehyde dehydrogenase, 3-HPDH, HIBADH, and4-hydroxybutyrate dehydrogenase activity. In certain embodiments, one ormore of the 3-HP pathway genes are exogenous, and in these embodimentsthe genes may be derived from a yeast, fungal, bacterial, plant, insect,or mammalian source.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from pyruvate to3-HP, wherein the cells contain one or more genes encoding enzymes withPDH, acetyl-CoA carboxylase, malonyl-CoA reductase, CoA acylatingmalonate semialdehyde dehydrogenase, 3-HPDH, HIBADH, and4-hydroxybutyrate dehydrogenase activity. In certain embodiments, one ormore of the 3-HP pathway genes are exogenous, and in these embodimentsthe genes may be derived from a yeast, fungal, bacterial, plant, insect,or mammalian source.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from pyruvate to3-HP, wherein the cells contain one or more genes encoding enzymes withalanine dehydrogenase, pyruvate/alanine aminotransferase, alanine 2,3aminomutase, CoA transferase, CoA synthetase, β-alanyl-CoA ammonialyase, 3-HP-CoA dehydratase, 3-HP-CoA hydrolase, 3-hydroxyisobutyryl-CoAhydrolase, BAAT, 3-HPDH, HIBADH, and 4-hydroxybutyrate dehydrogenaseactivity. In certain embodiments, one or more of the 3-HP pathway genesare exogenous, and in these embodiments the genes may be derived from ayeast, fungal, bacterial, plant, insect, or mammalian source.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from pyruvate to3-HP, wherein the cells contain one or more genes encoding enzymes withLDH, CoA transferase, lactyl-CoA dehydratase, 3-HP-CoA dehydratase,3-HP-CoA hydrolase, and 3-hydroxyisobutyryl-CoA hydrolase activity. Incertain embodiments, one or more of the 3-HP pathway genes areexogenous, and in these embodiments the genes may be derived from ayeast, fungal, bacterial, plant, insect, or mammalian source.

Provided herein in certain embodiments are genetically modified yeastcells comprising an active 3-HP fermentation pathway from PEP orpyruvate to 3-HP, wherein the cells contain one or more genes encodingenzymes with glycerol dehydratase and aldehyde dehydrogenase activity.In certain embodiments, one or more of the 3-HP pathway genes areexogenous, and in these embodiments the genes may be derived from ayeast, fungal, bacterial, plant, insect, or mammalian source.

The genetically modified yeast cells provided herein may be any yeastspecies. In certain embodiments, the cells are Crabtree-negative, and incertain of these embodiments they belong to the genus Issatchenkia,Candida, Kluyveromyces, Pichia, Schizosaccharomyces, Torulaspora,Zygosaccharomyces, or Saccharomyces. In certain of these embodiments,the cells may belong to the I. orientalis/P. fermentans clade or theSaccharomyces clade, and in these embodiments they may be I. orientalis,C. lambica, or S. bulderi. In certain embodiments, the yeast cells maybe 3-HP resistant yeast cells. 3-HP resistance may be a native trait ofthe cells or it result from the cells having undergone mutation and/orselection before, during, or after introduction of genetic modificationsrelated to an active 3-HP fermentation pathway, or a combinationthereof. In certain embodiments, the yeast cells may exhibit a degree oftolerance to organic acids other than 3-HP, other fermentation productsor byproducts, and/or various media components that is greater than thatexhibited by wild-type yeast cells of the same species. In certainembodiments, the yeast cells have undergone mutation and/or selection,such that the mutated and/or selected cells possess a higher degree ofresistance to 3-HP than a wild-type cell of the same species. In some ofthese embodiments, the cell has undergone mutation and/or selectionbefore being genetically modified with the one or more exogenous 3-HPpathway genes. In some embodiments, the cell has undergone selection inthe presence of lactic acid or 3-HP. In some embodiments, the selectionis chemostat selection.

In addition to modifications related to an active 3-HP fermentationpathway, the cells provided herein may contain deletions or disruptionsof one or more native genes. For example, the cells may containdeletions or disruptions of one or more PDC, ADH, GAL6, CYB2A, CYB2B,GPD, GPP, ALD, or PCK genes. In certain embodiments, these deletions ordisruptions may be coupled to the introduction of one or more genesrelated to an active 3-HP fermentation pathway.

Provided herein in certain embodiments are methods of producing 3-HPusing the genetically modified yeast cells provided herein by culturingthe cells in the presence of at least one carbon source and isolating3-HP from the culture medium. In certain of these embodiments, thecarbon source may be selected from one or more of glucose, xylose,arabinose, sucrose, fructose, cellulose, glucose oligomers, andglycerol.

BRIEF DESCRIPTION OF DRAWING

FIG. 1: Summary of select 3-HP fermentation pathways

FIG. 2: Plasmid pMIBa107

FIG. 3: Schematic representation of a targeted integration technique

FIG. 4: Plasmid pGMEr125(a)

FIG. 5: Plasmid pGMRr125(b)

FIG. 6: Plasmid pGMEr121

FIG. 7: Plasmid pMhCt074

FIG. 8: Plasmid pMhCt083

FIG. 9: Plasmid pMhCt087

FIG. 10: Plasmid pMhCt075

FIG. 11: Plasmid pMhCt077

FIG. 12: Plasmid pMhCt095

FIG. 13: Plasmid pMhCt096

FIG. 14: Plasmid pMeJi310-2

FIG. 15: Plasmid pMeJi312-2

FIG. 16: Plasmid pGMEr126

FIG. 17: Plasmid pGMEr130

FIG. 18: Plasmid pGMEr137

FIG. 19: Plasmid pACN5

FIG. 20: Plasmid pACN23

FIG. 21: Plasmid pHJJ27

FIG. 22: Plasmid pACN43

FIG. 23: Plasmid pHJJ75

FIG. 24: Plasmid pHJJ76

FIG. 25: Plasmid pJLJ49

FIG. 26: Plasmid pJLJ62

FIG. 27: Plasmid pM1458

FIG. 28: Plasmid pCM208

FIG. 29: Plasmid pJY39

FIG. 30: Plasmid pMcTs64

FIG. 31: Plasmid pMcTs65

FIG. 32: Plasmid pJLJ8.

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

All references cited herein are incorporated by reference in theirentirety.

Abbreviations

3-HP, 3-hydroxypropionic acid; 3-HPA, 3-hydroxypropionaldehyde; 3-HPDH,3-hydroxypropionic acid dehydrogenase; AAM, alanine 2,3 aminomutase;AAT, aspartate aminotransferase; ACC, acetyl-CoA carboxylase; ADC,aspartate 1-decarboxylase; AKG, alpha-ketoglutarate; ALD, aldehydedehydrogenase; BAAT, β-alanine aminotransferase; BCKA, branched-chainalpha-keto acid decarboxylase; bp, base pairs; CYB2,L-(+)-lactate-cytochrome c oxidoreductase; CYC, iso-2-cytochrome c; EMS,ethane methyl sulfonase; ENO, enolase; gabT, 4-aminobutyrateaminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase 3;GPD, glycerol 3-phosphate dehydrogenase; GPP, glycerol 3-phosphatephosphatase; HIBADH, 3-hydroxyisobutyrate dehydrogenase; IPDA,indolepyruvate decarboxylase; KGD, alpha-ketoglutarate decarboxylase;LDH, lactate dehydrogenase; MAE, malic enzyme; OAA, oxaloacetate; PCK,phosphoenolpyruvate carboxykinase; PDC, pyruvate decarboxylase; PDH,pyruvate dehydrogenase; PEP, phosphoenolpyruvate; PGK, phosphoglyceratekinase; PPC, phosphoenolpyruvate carboxylase; PYC, pyruvate carboxylase;RKI, ribose 5-phosphate ketol-isomerase; TAL, transaldolase; TEF1,translation elongation factor-1; TEF2, translation elongation factor-2;TKL, transketolase, XDH, xylitol dehydrogenase; XR, xylose reductase,YP, yeast extract/peptone.

Description

Provided herein are genetically modified yeast cells for the productionof 3-HP, methods of making these yeast cells, and methods of using thesecells to produce 3-HP. “3-HP” as used herein includes salt and acidforms of 3-hydroxypropionic acid.

A number of 3-HP fermentation pathways are known in the art (see, e.g.,U.S. Pat. No. 6,852,517; U.S. Pat. No. 7,309,597; US Pub. No.2001/0021978; US Pub. No. 2008/0199926; WO02/42418; and WO10/031083, allincorporated by reference herein). 3-HP fermentation pathways operatevia a series of intermediates that may include phosphoenolpyruvate(PEP), pyruvate, oxaloacetate (OAA), aspartate, β-alanine, malonatesemialdehyde, malate, malonyl-CoA, acetyl-CoA, alanine, lactate,lactyl-CoA, acrylyl-CoA, glycerol, 3-hydroxypropionaldehyde (3-HPA),β-alanyl-CoA, 3-HP-CoA, and glycerate. An overview of several of theknown 3-HP fermentation pathways is set forth in FIG. 1.

As disclosed herein, a set of yeast cells from various species weretested for 3-HP resistance. Cells exhibiting 3-HP resistance werefurther evaluated based on their growth rates and glucose consumptionrates in media containing varying concentrations of 3-HP. Based on theseexperiments, a set of ideal host cells for 3-HP production wereidentified. These host cells were then genetically modified to containan active 3-HP fermentation pathway, resulting in genetically modifiedyeast cells that produce 3-HP under low pH conditions.

Provided herein in certain embodiments are genetically modified yeastcells having at least one active 3-HP fermentation pathway from PEP,pyruvate, and/or glycerol to 3-HP. A yeast cell having an “active 3-HPfermentation pathway” as used herein produces active enzymes necessaryto catalyze each reaction in a 3-HP fermentation pathway, and thereforeis capable of producing 3-HP in measurable yields when cultured underfermentation conditions in the presence of at least one fermentablesugar. A yeast cell having an active 3-HP fermentation pathway comprisesone or more 3-HP pathway genes. A “3-HP pathway gene” as used hereinrefers to the coding region of a nucleotide sequence that encodes anenzyme involved in a 3-HP fermentation pathway.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through PEP or pyruvate, OAA,aspartate, β-alanine, and malonate semialdehyde intermediates (see,e.g., US Pub. No. 2010/0021978, FIG. 1). In these embodiments, the yeastcells comprise a set of 3-HP fermentation pathway genes comprising oneor more of pyruvate carboxylase (PYC), PEP carboxylase (PPC), aspartateaminotransferase (AAT), aspartate 1-decarboxylase (ADC), β-alanineaminotransferase (BAAT), aminobutyrate aminotransferase (gabT), 3-HPdehydrogenase (3-HPDH), 3-hydroxyisobutyrate dehydrogenase (HIBADH), and4-hydroxybutyrate dehydrogenase genes. The 3-HP fermentation pathwaygenes may also include a PEP carboxykinase (PCK) gene that has beenmodified to produce a polypeptide that preferably catalyzes theconversion of PEP to OAA (native PCK genes generally produce apolypeptide that preferably catalyzes the reverse reaction of OAA toPEP).

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through PEP or pyruvate, OAA,and malate intermediates (see, e.g., US Pub. No. 2010/0021978, FIG. 4).In these embodiments, the yeast cells comprise a set of 3-HPfermentation pathway genes comprising one or more of PPC, PYC, malatedehydrogenase, and malate decarboxylase genes. The 3-HP fermentationpathway genes may also include a PCK gene that has been modified toproduce a polypeptide that preferably catalyzes the conversion of PEP toOAA.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through PEP or pyruvate, OAA,and malonate semialdehyde intermediates (see, e.g., US Pub. No.2010/0021978, FIG. 1). In these embodiments, the yeast cells comprise aset of 3-HP fermentation pathway genes comprising one or more of PPC,PYC, 2-keto acid decarboxylase, alpha-ketoglutarate (AKG) decarboxylase(KGD), branched-chain alpha-keto acid decarboxylase (BCKA),indolepyruvate decarboxylase (IPDA), 3-HPDH, HIBADH, and4-hydroxybutyrate dehydrogenase genes. The 3-HP fermentation pathwaygenes may also include a PCK gene that has been modified to produce apolypeptide that preferably catalyzes the conversion of PEP to OAA.Further, the 3-HP fermentation pathway genes may include a PDC geneand/or benzoylformate decarboxylase gene that has been modified toencode a polypeptide capable of catalyzing the conversion of OAA tomalonate semialdehyde.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through PEP or pyruvate, OAA,malonyl-CoA, and malonate semialdehyde intermediates, wherein themalonate semialdehyde intermediate is optional (see, e.g., US Pub. No.2010/0021978, FIG. 2). In these embodiments, the yeast cells comprise aset of 3-HP fermentation pathway genes comprising one or more of PPC,PYC, OAA formatelyase, malonyl-CoA reductase, CoA acylating malonatesemialdehyde dehydrogenase, 3-HPDH, HIBADH, and 4-hydroxybutyratedehydrogenase genes. The 3-HP fermentation pathway genes may alsoinclude a PCK gene that has been modified to produce a polypeptide thatpreferably catalyzes the conversion of PEP to OAA. Further, the 3-HPfermentation pathway genes may include an OAA dehydrogenase gene derivedby modifying a 2-keto-acid dehydrogenase gene to produce a polypeptidethat catalyzes the conversion of OAA to malonyl-CoA.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through pyruvate, acetyl-CoA,malonyl-CoA, and malonate semialdehyde intermediates, wherein themalonate semialdehyde intermediate is optional (see, e.g., WO02/042418,FIG. 44). In these embodiments, the yeast cells comprise a set of 3-HPfermentation pathway genes comprising one or more of pyruvatedehydrogenase (PDH), acetyl-CoA carboxylase (ACC), malonyl-CoAreductase, CoA acylating malonate semialdehyde dehydrogenase, 3-HPDH,HIBADH, and 4-hydroxybutyrate dehydrogenase genes.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through pyruvate, alanine,β-alanine, β-alanyl-CoA, acrylyl-CoA, 3-HP-CoA, and malonatesemialdehyde intermediates, wherein the β-alanyl-CoA, acrylyl-CoA,3-HP-CoA, and malonate semialdehyde intermediates are optional(β-alanine can be converted to 3-HP via a malonate semialdehydeintermediate or via β-alanyl-CoA, acrylyl-CoA, and 3-HP-CoAintermediates (see, e.g., U.S. Pat. No. 7,309,597, FIG. 1). In theseembodiments, the yeast cells comprise a set of 3-HP fermentation pathwaygenes comprising one or more of alanine dehydrogenase, pyruvate/alanineaminotransferase, alanine 2,3 aminomutase, CoA transferase, CoAsynthetase, β-alanyl-CoA ammonia lyase, 3-HP-CoA dehydratase, 3-HP-CoAhydrolase, 3-hydroxyisobutyryl-CoA hydrolase, BAAT, 3-HPDH, HIBADH, and4-hydroxybutyrate dehydrogenase genes.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through pyruvate, lactate,lactyl-CoA, acrylyl-CoA, and 3-HP-CoA intermediates (see, e.g.,WO02/042418, FIG. 1). In these embodiments, the yeast cells comprise aset of 3-HP fermentation pathway genes comprising one or more of LDH,CoA transferase, CoA synthetase, lactyl-CoA dehydratase, 3-HP-CoAdehydratase, 3-HP-CoA hydrolase, and 3-hydroxyisobutyryl-CoA hydrolasegenes.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through glycerol and 3-HPAintermediates (see, e.g., U.S. Pat. No. 6,852,517). In theseembodiments, the yeast cells comprise a set of 3-HP fermentation pathwaygenes comprising one or more of glycerol dehydratase and aldehydedehydrogenase genes.

In certain embodiments, the yeast cells provided herein have an active3-HP fermentation pathway that proceeds through PEP or pyruvate, OAA,aspartate, β-alanine, β-alanyl-CoA, acrylyl-CoA, 3-HP-CoA, and alanineintermediates, wherein the OAA, aspartate, and alanine intermediates areoptional (PEP or pyruvate can be converted to β-alanine via OAA andaspartate or via alanine) (see WO02/042418, FIG. 54; U.S. Pat. No.7,309,597, FIG. 1). In these embodiments, the yeast cells comprise a setof 3-HP fermentation pathway genes comprising one or more of PPC, PYC,AAT, ADC, CoA transferase, CoA synthetase, β-alanyl-CoA ammonia lyase,3-HP-CoA dehydratase, 3-HP-CoA hydrolase, 3-hydroxyisobutyrl-CoAhydrolase, alanine dehydrogenase, pyruvate/alanine aminotransferase, andAAM genes. The 3-HP fermentation pathway genes may also include a PCKgene that has been modified to produce a polypeptide that preferablycatalyzes the conversion of PEP to OAA.

The 3-HP fermentation pathway genes in the yeast cells provided hereinmay be endogenous or exogenous. “Endogenous” as used herein with regardto genetic components such as genes, promoters, and terminator sequencesmeans that the genetic component is present at a particular location inthe genome of a native form of a particular yeast cell. “Exogenous” asused herein with regard to genetic components means that the geneticcomponent is not present at a particular location in the genome of anative form of a particular yeast cell. “Native” as used herein withregard to a yeast cell refers to a wild-type yeast cell of a particularyeast species. “Native” as used herein with regard to a metabolicpathway refers to a metabolic pathway that exists and is active in anative yeast cell.

An exogenous genetic component may have either a native or non-nativesequence. An exogenous genetic component with a native sequencecomprises a sequence identical to (apart from individual-to-individualmutations which do not affect function) a genetic component that ispresent in the genome of a native cell (i.e., the exogenous geneticcomponent is identical to an endogenous genetic component). However, theexogenous component is present at a different location in the host cellgenome than the endogenous component. For example, an exogenous PYC genethat is identical to an endogenous PYC gene may be inserted into a yeastcell, resulting in a modified cell with a non-native (increased) numberof PYC gene copies. An exogenous genetic component with a non-nativesequence comprises a sequence that is not found in the genome of anative cell. For example, an exogenous PYC gene from a particularspecies may be inserted into a yeast cell of another species. Anexogenous gene is preferably integrated into the host cell genome in afunctional manner, meaning that it is capable of producing an activeprotein in the host cell. However, in certain embodiments the exogenousgene may be introduced into the cell as part of a vector that is stablymaintained in the host cytoplasm.

In certain embodiments, the yeast cells provided herein comprise one ormore exogenous 3-HP fermentation pathway genes. In certain embodiments,the genetically modified yeast cells disclosed herein comprise a singleexogenous gene. In other embodiments, the yeast cells comprise multipleexogenous genes. In these embodiments, the yeast cells may comprisemultiple copies of a single exogenous gene and/or copies of two or moredifferent exogenous genes. Yeast cells comprising multiple exogenousgenes may comprise any number of exogenous genes. For example, theseyeast cells may comprise 1 to 20 exogenous genes, and in certainpreferred embodiments they may comprise 1 to 7 exogenous genes. Multiplecopies of an exogenous gene may be integrated at a single locus suchthat they are adjacent to one another. Alternatively, they may beintegrated at several loci within the host cell's genome.

In certain embodiments, the yeast cells provided herein comprise one ormore endogenous 3-HP fermentation pathway genes. In certain of theseembodiments, the cells may be engineered to overexpress one or more ofthese endogenous genes, meaning that the modified cells express theendogenous gene at a higher level than a native cell under at least someconditions. In certain of these embodiments, the endogenous gene beingoverexpressed may be operatively linked to one or more exogenousregulatory elements. For example, one or more native or non-nativeexogenous strong promoters may be introduced into a cell such that theyare operatively linked to one or more endogenous 3-HP pathway genes.

3-HP fermentation pathway genes in the modified yeast cells providedherein may be operatively linked to one or more regulatory elements suchas a promoter or terminator. As used herein, the term “promoter” refersto an untranslated sequence located upstream (i.e., 5′) to thetranslation start codon of a gene (generally within about 1 to 1000 basepairs (bp), preferably within about 1 to 500 bp) which controls thestart of transcription of the gene. The term “terminator” as used hereinrefers to an untranslated sequence located downstream (i.e., 3′) to thetranslation finish codon of a gene (generally within about 1 to 1000 bp,preferably within about 1 to 500 bp, and especially within about 1 to100 bp) which controls the end of transcription of the gene. A promoteror terminator is “operatively linked” to a gene if its position in thegenome relative to that of the gene is such that the promoter orterminator, as the case may be, performs its transcriptional controlfunction. Suitable promoters and terminators are described, for example,in WO99/14335, WO00/71738, WO02/42471, WO03/102201, WO03/102152 andWO03/049525 (all incorporated by reference herein in their entirety).

Regulatory elements linked to 3-HP fermentation pathway genes in thecells provided herein may be endogenous or exogenous. For example, anexogenous 3-HP fermentation pathway gene may be inserted into a yeastcell such that it is under the transcriptional control of an endogenouspromoter and/or terminator. Alternatively, the exogenous 3-HPfermentation pathway gene may be linked to one or more exogenousregulatory elements. For example, an exogenous gene may be introducedinto the cell as part of a gene expression construct that comprises oneor more exogenous regulatory elements. In certain embodiments, exogenousregulatory elements, or at least the functional portions of exogenousregulatory elements, may comprise native sequences. In otherembodiments, exogenous regulatory elements may comprise non-nativesequences. In these embodiments, the exogenous regulatory elements maycomprise a sequence with a relatively high degree of sequence identityto a native regulatory element. For example, an exogenous gene may belinked to an exogenous promoter or terminator having at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% sequence identityto a native promoter or terminator. Sequence identity percentages fornucleotide or amino acid sequences can be calculated by methods known inthe art, such as for example using BLAST (National Center for BiologicalInformation (NCBI) Basic Local Alignment Search Tool) version 2.2.1software with default parameters. For example, a sequences having anidentity score of at least 90%, using the BLAST version 2.2.1 algorithmwith default parameters is considered to have at least 90% sequenceidentity. The BLAST software is available from the NCBI, Bethesda, Md.

In certain aspects, a regulatory element (e.g., a promoter) linked to a3-HP fermentation pathway gene in the cells provided herein may beforeign to the pathway gene. A regulatory element that is foreign to apathway gene is a regulatory element that is not liked to the gene inits natural form. The skilled artisan can appreciate that a regulatoryelement foreign to a pathway gene can be endogenous or exogenous,depending on the pathway gene and its relation to the yeast cell. Insome instances, an endogenous 3-HP fermentation pathway gene isoperatively linked to a regulatory element (e.g., a promoter) that isforeign to the pathway gene. In other instances, an exogenous 3-HPfermentation pathway gene is operatively linked to an exogenousregulatory element (e.g., a promoter) that is foreign to the pathwaygene.

In those embodiments wherein multiple exogenous genes are inserted intoa host cell, each exogenous gene may be under the control of a differentregulatory element, or two or more exogenous genes may be under thecontrol of the same regulatory elements. For example, where a firstexogenous gene is linked to a first regulatory element, a secondexogenous gene may also be linked to the first regulatory element, or itmay be linked to a second regulatory element. The first and secondregulatory elements may be identical or share a high degree of sequenceidentity, or they be wholly unrelated.

Examples of promoters that may be linked to one or more 3-HPfermentation pathway genes in the yeast cells provided herein include,but are not limited to, promoters for PDC1, phosphoglycerate kinase(PGK), xylose reductase (XR), xylitol dehydrogenase (XDH),L-(+)-lactate-cytochrome c oxidoreductase (CYB2), translation elongationfactor-1 (TEF1), translation elongation factor-2 (TEF2), enolase (ENO1),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and orotidine5′-phosphate decarboxylase (URA3) genes. In these examples, the 3-HPfermentation pathway genes may be linked to endogenous or exogenouspromoters for PDC1, PGK, XR, XDH, CYB2, TEF1, TEF2, ENO1, GAPDH, or URA3genes. Where the promoters are exogenous, they may be identical to orshare a high degree of sequence identity (i.e., at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at leastabout 99%) with native promoters for PDC1, PGK, XR, XDH, CYB2, TEF1,TEF2, ENO1, GAPDH, or URA3 genes.

Examples of terminators that may be linked to one or more 3-HPfermentation pathway genes in the yeast cells provided herein include,but are not limited to, terminators for PDC1, XR, XDH, transaldolase(TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI),CYB2, or iso-2-cytochrome c (CYC) genes or the galactose family of genes(especially the GAL10 terminator). In these examples, the 3-HPfermentation pathway genes may be linked to endogenous or exogenousterminators for PDC1, XR, XDH, TAL, TKL, RKI, CYB2, or CYC genes orgalactose family genes. Where the terminators are exogenous, they may beidentical to or share a high degree of sequence identity (i.e., at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or at least about 99%) with native terminators for PDC1, XR, XDH, TAL,TKL, RKI, CYB2, or CYC genes or galactose family genes. In certainembodiments, 3-HP fermentation pathway genes are linked to a terminatorthat comprises a functional portion of a native GAL10 gene native to thehost cell or a sequence that shares at least 80%, at least 85%, at least90%, or at least 95% sequence identity with a native GAL10 terminator.

Exogenous genes may be inserted into a yeast host cell via any methodknown in the art. In preferred embodiments, the genes are integratedinto the host cell genome. Exogenous genes may be integrated into thegenome in a targeted or a random manner. In those embodiments where thegene is integrated in a targeted manner, it may be integrated into theloci for a particular gene, such that integration of the exogenous geneis coupled to deletion or disruption of a native gene. For example,introduction of an exogenous 3-HP pathway gene may be coupled todeletion or disruption of one or more genes encoding enzymes involved inother fermentation product pathways. Alternatively, the exogenous genemay be integrated into a portion of the genome that does not correspondto a gene.

Targeted integration and/or deletion may utilize an integrationconstruct. The term “construct” as used herein refers to a DNA sequencethat is used to transform a host cell. The construct may be, forexample, a circular plasmid or vector, a portion of a circular plasmidor vector (such as a restriction enzyme digestion product), a linearizedplasmid or vector, or a PCR product prepared using a plasmid or genomicDNA as a template. Methods for transforming a yeast cell with anexogenous construct are described in, for example, WO99/14335,WO00/71738, WO02/42471, WO03/102201, WO03/102152, and WO03/049525. Anintegration construct can be assembled using two cloned target DNAsequences from an insertion site target. The two target DNA sequencesmay be contiguous or non-contiguous in the native host genome. In thiscontext, “non-contiguous” means that the DNA sequences are notimmediately adjacent to one another in the native genome, but areinstead are separated by a region that is to be deleted. “Contiguous”sequences as used herein are directly adjacent to one another in thenative genome. Where targeted integration is to be coupled to deletionor disruption of a target gene, the integration construct may also bereferred to as a deletion construct. In a deletion construct, one of thetarget sequences may include a region 5′ to the promoter of the targetgene, all or a portion of the promoter region, all or a portion of thetarget gene coding sequence, or some combination thereof. The othertarget sequence may include a region 3′ to the terminator of the targetgene, all or a portion of the terminator region, and/or all or a portionof the target gene coding sequence. Where targeted integration is not tobe coupled to deletion or disruption of a native gene, the targetsequences are selected such that insertion of an intervening sequencewill not disrupt native gene expression. An integration or deletionconstruct is prepared such that the two target sequences are oriented inthe same direction in relation to one another as they natively appear inthe genome of the host cell. Where an integration or deletion constructis used to introduce an exogenous gene into a host cell, a geneexpression cassette is cloned into the construct between the two targetgene sequences to allow for expression of the exogenous gene. The geneexpression cassette contains the exogenous gene, and may further includeone or more regulatory sequences such as promoters or terminatorsoperatively linked to the exogenous gene. Deletion constructs can alsobe constructed that do not contain a gene expression cassette. Suchconstructs are designed to delete or disrupt a gene sequence without theinsertion of an exogenous gene.

An integration or deletion construct may comprise one or more selectionmarker cassettes cloned into the construct between the two target genesequences. The selection marker cassette contains at least one selectionmarker gene that allows for selection of transformants. A “selectionmarker gene” is a gene that encodes a protein needed for the survivaland/or growth of the transformed cell in a selective culture medium, andtherefore can be used to apply selection pressure to the cell.Successful transformants will contain the selection marker gene, whichimparts to the successfully transformed cell at least one characteristicthat provides a basis for selection. Typical selection marker genesencode proteins that (a) confer resistance to antibiotics or othertoxins (e.g., resistance to bleomycin or zeomycin (e.g.,Streptoalloteichus hindustanus ble gene), aminoglycosides such as G418or kanamycin (e.g., kanamycin resistance gene from transposon Tn903), orhygromycin (e.g., aminoglycoside antibiotic resistance gene from E.coli)), (b) complement auxotrophic deficiencies of the cell (e.g.,deficiencies in leucine (e.g., K. marxianus LEU2 gene), uracil (e.g., K.marxianus, S. cerevisiae, or I. orientalis URA3 gene), or tryptophan(e.g., K. marxianus, S. cerevisiae, or I. orientalis TRP gene)), (c)enable the cell to synthesize critical nutrients not available fromsimple media, or (d) confer the ability for the cell to grow on aparticular carbon source (e.g., MEL5 gene from S. cerevisiae, whichencodes the alpha-galactosidase (melibiase) enzyme and confers theability to grow on melibiose as the sole carbon source). Preferredselection markers include the URA3 gene, zeocin resistance gene, G418resistance gene, MEL5 gene, and hygromycin resistance gene. Anotherpreferred selection marker is an L-lactate:ferricytochrome coxidoreductase (CYB2) gene cassette, provided that the host cell eithernatively lacks such a gene or that its native CYB2 gene(s) are firstdeleted or disrupted. A selection marker gene is operatively linked toone or more promoter and/or terminator sequences that are operable inthe host cell. In certain embodiments, these promoter and/or terminatorsequences are exogenous promoter and/or terminator sequences that areincluded in the selection marker cassette. Suitable promoters andterminators are as described herein.

An integration or deletion construct is used to transform the host cell.Transformation may be accomplished using, for example, electroporationand/or chemical transformation (e.g., calcium chloride, lithiumacetate-based, etc.) methods. Selection or screening based on thepresence or absence of the selection marker may be performed to identifysuccessful transformants. In successful transformants, homologousrecombination events at the locus of the target site results in thedisruption or the deletion of the target site sequence. Where theconstruct targets a native gene for deletion or disruption, all or aportion of the native target gene, its promoter, and/or its terminatormay be deleted during this recombination event. The expression cassette,selection marker cassette, and any other genetic material between thetarget sequences in the integration construct is inserted into the hostgenome at the locus corresponding to the target sequences. Analysis byPCR or Southern analysis can be performed to confirm that the desiredinsertion/deletion has taken place.

In some embodiments, cell transformation may be performed using DNA fromtwo or more constructs, PCR products, or a combination thereof, ratherthan a single construct or PCR product. In these embodiments, the 3′ endof one integration fragment overlaps with the 5′ end of anotherintegration fragment. In one example, one construct will contain thefirst sequence from the locus of the target sequence and anon-functional part of the marker gene cassette, while the other willcontain the second sequence from the locus of the target sequence and asecond non-functional part of the marker gene cassette. The parts of themarker gene cassette are selected such that they can be combined to forma complete cassette. The cell is transformed with these piecessimultaneously, resulting in the formation of a complete, functionalmarker or structural gene cassette. Successful transformants can beselected for on the basis of the characteristic imparted by theselection marker. In another example, the selection marker resides onone fragment but the target sequences are on separate fragments, so thatthe integration fragments have a high probability of integrating at thesite of interest. In other embodiments, transformation from three linearDNAs can be used to integrate exogenous genetic material. In theseembodiments, one fragment overlaps on the 5′ end with a second fragmentand on the 3′ end with a third fragment.

An integration or deletion construct may be designed such that theselection marker gene and some or all of its regulatory elements canbecome spontaneously deleted as a result of a subsequent homologousrecombination event. A convenient way of accomplishing this is to designthe construct such that the selection marker gene and/or regulatoryelements are flanked by repeat sequences. Repeat sequences are identicalDNA sequences, native or non-native to the host cell, and oriented onthe construct in the same or opposite direction with respect to oneanother. The repeat sequences are advantageously about 50 to 1500 bp inlength, and do not have to encode for anything. Inclusion of the repeatsequences permits a homologous recombination event to occur, whichresults in deletion of the selection marker gene and one of the repeatsequences. Since homologous recombination occurs with relatively lowfrequency, it may be necessary to grow transformants for several roundson nonselective media to allow for the spontaneous homologousrecombination to occur in some of the cells. Cells in which theselection marker gene has become spontaneously deleted can be selectedor screened on the basis of their loss of the selection characteristicimparted by the selection marker gene. In certain cases, expression of arecombinase enzyme may enhance recombination between the repeated sites.

An exogenous 3-HP fermentation pathway gene in the modified yeast cellsprovided herein may be derived from a source gene from any suitablesource. For example, an exogenous gene may be derived from a yeast,fungal, bacterial, plant, insect, or mammalian source. As used herein,an exogenous gene that is “derived from” a native source gene encodes apolypeptide that 1) is identical to a polypeptide encoded by the nativegene, 2) shares at least 50%, at least 60%, at least 70%, at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity with a polypeptide encoded by the native gene, and/or3) has the same function in a 3-HP fermentation pathway as thepolypeptide encoded by the native gene. For example, a PYC gene that isderived from a I. orientalis PYC gene may encode a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2, a polypeptide withat least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the amino acid sequence of SEQ ID NO: 2, and/or a polypeptide thathas the ability to catalyze the conversion of pyruvate to OAA. A genederived from a native gene may comprise a nucleotide sequence with atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the coding region of the native gene. In certain embodiments, a genederived from a native gene may comprise a nucleotide sequence that isidentical to the coding region of the source gene. For example, a PYCgene that is derived from a I. orientalis PYC gene may comprise thenucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence with atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the nucleotide sequence of SEQ ID NO: 1.

In certain embodiments of the modified yeast cells provided herein, thenative source gene from which the exogenous 3-HP fermentation pathwaygene is derived produces a polypeptide that is involved in a 3-HPfermentation pathway. In other embodiments, however, the native sourcegene may encode a polypeptide that is not involved in a 3-HPfermentation pathway or that catalyzes a reverse reaction in a 3-HPfermentation pathway. In these embodiments, the exogenous 3-HP pathwaygene will have undergone one or more targeted or random mutations versusthe native source gene that result in modified activity and/or substratepreference. For example, a native source gene may be mutated to generatea gene that encodes a polypeptide with increased activity in a desiredreaction direction and/or decreased activity in a non-desired directionin a 3-HP fermentation pathway. For example, where the native sourcegene encodes a polypeptide capable of catalyzing both a forward andreverse reactions in a 3-HP fermentation pathway, the gene may bemodified such that the resultant exogenous gene has increased activityin the forward direction and decreased activity in the reversedirection. Similarly, a native source gene may be mutated to produce agene that encodes a polypeptide with different substrate preference thanthe native polypeptide. For example, a 3-HP pathway gene may be mutatedto produce a polypeptide with the ability to act on a substrate that iseither not preferred or not acted on at all by the native polypeptide.In these embodiments, the polypeptide encoded by the exogenous 3-HPpathway gene may catalyze a reaction that the polypeptide encoded by thenative source gene is completely incapable of catalyzing. A nativesource gene may also be mutated such that the resultant 3-HP pathwaygene exhibits decreased feedback inhibition at the DNA, RNA, or proteinlevel in the presence of one or more downstream 3-HP pathwayintermediates or side products.

In certain embodiments of the modified yeast cells provided herein, anexogenous 3-HP pathway gene may be derived from the host yeast species.For example, where the host cell is I. orientalis, an exogenous gene maybe derived from an I. orientalis gene. In these embodiments, theexogenous gene may comprise a nudeotide sequence identical to the codingregion of the native gene, such that incorporation of the exogenous geneinto the host cell increases the copy number of a native gene sequenceand/or changes the regulation or expression level of the gene if underthe control of a promoter that is different from the promoter thatdrives expression of the gene in a wild-type cell. In other embodiments,the exogenous 3-HP pathway gene may comprise a nucleotide sequence thatdiffers from the coding region of a native 3-HP pathway gene, butnonetheless encodes a polypeptide that is identical to the polypeptideencoded by the native 3-HP pathway gene. In still other embodiments, theexogenous 3-HP pathway gene may comprise a nucleotide sequence thatencodes a polypeptide with at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to a polypeptide encoded by one or morenative 3-HP pathway genes. In certain of these embodiments, theexogenous gene comprises a nucleotide sequence with at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% sequence identity to one ormore native genes. In still other embodiments, the exogenous 3-HP genemay encode a polypeptide that has less than 50% sequence identity to apolypeptide encoded by a native 3-HP pathway gene but which nonethelesshas the same function as the native polypeptide in a 3-HP fermentationpathway (i.e., the ability to catalyze the same reaction). A nativesource gene may be subjected to mutagenesis if necessary to provide acoding sequence starting with the usual eukaryotic starting codon (ATG),or for other purposes.

In other embodiments, the exogenous 3-HP pathway gene may be derivedfrom a species that is different than that of the host yeast cell. Incertain of these embodiments, the exogenous 3-HP pathway gene may bederived from a different yeast species than the host cell. For example,where the host cell is I. orientalis, the exogenous gene may be derivedfrom S. cerevisiae. In other embodiments, the exogenous 3-HP pathwaygene may be derived from a fungal, bacterial, plant, insect, ormammalian source. For example, where the host cell is I. orientalis, theexogenous gene may be derived from a bacterial source such as E. coli.In those embodiments where the exogenous 3-HP pathway gene is derivedfrom a non-yeast source, the exogenous gene sequence may becodon-optimized for expression in a yeast host cell.

In those embodiments where the exogenous 3-HP pathway gene is derivedfrom a species other than the host cell species, the exogenous gene mayencode a polypeptide identical to a polypeptide encoded by a native 3-HPpathway gene from the source organism. In certain of these embodiments,the exogenous 3-HP pathway gene may be identical to a native 3-HPpathway gene from the source organism. In other embodiments, theexogenous gene may share at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to a native 3-HP pathway gene from thesource organism. In other embodiments, the exogenous 3-HP pathway genemay encode a polypeptide that shares at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, or at least 99% sequence identity with a polypeptide encodedby a native 3-HP pathway gene from the source organism. In certain ofthese embodiments, the exogenous gene may comprise a nucleotide sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to one or more native 3-HP pathway genes from the sourceorganism. In still other embodiments, the exogenous 3-HP gene may encodea polypeptide that has less than 50% sequence identity to a polypeptideencoded by a native 3-HP pathway gene from the source organism, butwhich nonetheless has the same function as the native polypeptide fromthe source organism in a 3-HP fermentation pathway.

In certain embodiments, the yeast cells provided herein express one ormore 3-HP pathway genes encoding enzymes selected from the groupconsisting of ACC (catalyzes the conversion of acetyl-CoA tomalonyl-CoA), alanine 2,3 aminomutase (AAM, catalyzes the conversion ofalanine to β-alanine), alanine dehydrogenase (catalyzes the conversionof pyruvate to alanine), aldehyde dehydrogenase (catalyzes theconversion of 3-HPA to 3-HP), KGD (catalyzes the conversion of OAA tomalonate semialdehyde), AAT (catalyzes the conversion of OAA toaspartate), ADC (catalyzes the conversion of aspartate to β-alanine),BCKA (catalyzes the conversion of OAA to malonate semialdehyde), BAAT(catalyzes the conversion of β-alanine to malonate semialdehyde),4-aminobutyrate aminotransferase (gabT, catalyzes the conversion ofβ-alanine to malonate semialdehyde), β-alanyl-CoA ammonia lyase(catalyzes the conversion of β-alanyl-CoA to acrylyl-CoA), Co-Aacylating malonate semialdehyde dehydrogenase (catalyzes the conversionof malonyl-CoA to malonate semialdehyde), CoA synthetase (catalyzes theconversion of β-alanine to β-alanyl-CoA or the conversion of lactate tolactyl-CoA), CoA transferase (catalyzes the conversion of β-alanine toβ-alanyl-CoA and/or the conversion of lactate to lactyl-CoA), glyceroldehydratase (catalyzes the conversion of glycerol to 3-HPA), IPDA(catalyzes the conversion of OAA to malonate semialdehyde), LDH(catalyzes the conversion of pyruvate to lactate), lactyl-CoAdehydratase (catalyzes the conversion of lactyl-CoA to acrylyl-CoA),malate decarboxylase (catalyzes the conversion of malate to 3-HP),malate dehydrogenase (catalyzes the conversion of OAA to malate),malonyl-CoA reductase (catalyzes the conversion of malonyl-CoA tomalonate semialdehyde or 3-HP), OAA formatelyase (also known aspyruvate-formate lyase and ketoacid formate-lyase, catalyzes theconversion of OAA to malonyl-CoA), OAA dehydrogenase (catalyzes theconversion of OAA to malonyl CoA); PPC (catalyzes the conversion of PEPto OAA), pyruvate/alanine aminotransferase (catalyzes the conversion ofpyruvate to alanine), PYC (catalyzes the conversion of pyruvate to OAA),PDH (catalyzes the conversion of pyruvate to acetyl-CoA), 2-keto aciddecarboxylase (catalyzes the conversion of OAA to malonatesemialdehyde), 3-HP-CoA dehydratase (also known as acrylyl-CoAhydratase, catalyzes the conversion of acrylyl-CoA to 3-HP-CoA), 3-HPDH(catalyzes the conversion of malonate semialdehyde to 3-HP), 3-HP-CoAhydrolase (catalyzes the conversion of 3-HP-CoA to 3-HP), HIBADH(catalyzes the conversion of malonate semialdehyde to 3-HP),3-hydroxyisobutyryl-CoA hydrolase (catalyzes the conversion of 3-HP-CoAto 3-HP), and 4-hydroxybutyrate dehydrogenase (catalyzes the conversionof malonate semialdehyde to 3-HP). For each of these enzyme activities,the reaction of interest in parentheses may be a result of native ornon-native activity.

A “pyruvate carboxylase gene” or “PYC gene” as used herein refers to anygene that encodes a polypeptide with pyruvate carboxylase activity,meaning the ability to catalyze the conversion of pyruvate, CO₂, and ATPto OAA, ADP, and phosphate. In certain embodiments, a PYC gene may bederived from a yeast source. For example, the PYC gene may be derivedfrom an I. orientalis PYC gene encoding the amino acid sequence setforth in SEQ ID NO: 2. In other embodiments, the gene may encode anamino acid sequence with at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, an I. orientalis-derived PYC gene may comprisethe nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotidesequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1.In other embodiments, the PYC gene may be derived from a bacterialsource. For example, the PYC gene may be derived from one of the fewbacterial species that use only PYC and not PPC (see below) foranaplerosis, such as R. sphaeroides, or from a bacterial species thatpossesses both PYC and PPC, such as R. etli. The amino acid sequencesencoded by the PYC genes of R. sphaeroides and R. etli are set forth inSEQ ID NOs: 3 and 4, respectively. A PYC gene may be derived from a geneencoding the amino acid sequence of SEQ ID NOs: 3 or 4, or from a geneencoding an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NOs: 3 or 4. Alternatively, the PYC gene may be derived from aPYC gene encoding an enzyme that does not have a dependence onacetyl-CoA for activation, such as a P. fluorescens PYC gene encodingthe amino acid sequence set forth in SEQ ID NO: 5 (carboxytransferasesubunit) or SEQ ID NO: 6 (biotin carboxylase subunit), a C. glutamicumPYC gene of encoding the amino acid sequence set forth in SEQ ID NO: 7,or a gene encoding an amino acid sequence with at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99% sequence identity to the amino acidsequence of SEQ ID NOs: 5, 6, or 7. A PYC gene may also be derived froma PYC gene that encodes an enzyme that is not inhibited by aspartate,such as an S. meliloti PYC gene encoding the amino acid sequence setforth in SEQ ID NO: 8 (Sauer FEMS Microbiol Rev 29:765 (2005), or from agene encoding an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO: 8.

A “PEP carboxylase gene” or “PPC gene” as used herein refers to any genethat encodes a polypeptide with PEP carboxylase activity, meaning theability to catalyze the conversion of PEP and CO₂ to OAA and phosphate.In certain embodiments, a PPC gene may be derived from a bacterial PPCgene. For example, the PPC gene may be derived from an E. coli PPC geneencoding the amino acid sequence set forth in SEQ ID NO: 10 or an aminoacid sequence with at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least99% sequence identity to the amino acid sequence of SEQ ID NO: 10. Incertain embodiments, an E. coli-derived PPC gene may comprise thenucleotide sequence set forth in SEQ ID NO: 9 or a nucleotide sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 9. In otherembodiments, a PPC gene may be derived from an “A” type PPC, found inmany archea and a limited number of bacteria, that is not activated byacetyl CoA and is less inhibited by aspartate. For example, a PPC genemay be derived from an M. thermoautotrophicum PPC A gene encoding theamino acid sequence set forth in SEQ ID NO: 11, a C. perfringens PPC Agene encoding the amino acid sequence set forth in SEQ ID NO: 12, or agene encoding an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NOs: 11 or 12. In certain of these embodiments, the gene mayhave undergone one or more mutations versus the native gene in order togenerate an enzyme with improved characteristics. For example, the genemay have been mutated to encode a PPC polypeptide with increasedresistance to aspartate feedback versus the native polypeptide. In otherembodiments, the PPC gene may be derived from a plant source.

An “aspartate aminotransferase gene” or “AAT gene” as used herein refersto any gene that encodes a polypeptide with aspartate aminotransferaseactivity, meaning the ability to catalyze the conversion of OAA toaspartate. Enzymes having aspartate aminotransferase activity areclassified as EC 2.6.1.1. In certain embodiments, an AAT gene may bederived from a yeast source such as I. orientalis or S. cerevisiae. Forexample, the AAT gene may be derived from an I. orientalis AAT geneencoding the amino acid sequence set forth in SEQ ID NO: 14 or an S.cerevisiae AAT2 gene encoding the amino acid sequence set forth in SEQID NO: 15. In other embodiments, the gene may encode an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NOs: 14 or 15. Incertain embodiments, an I. orientalis-derived AAT gene may comprise thenucleotide sequence set forth in SEQ ID NO: 13 or a nucleotide sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 13. In otherembodiments, the AAT gene may be derived from a bacterial source. Forexample, the AAT gene may be derived from an E. coli aspC gene encodinga polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16. In other embodiments, the gene may encode an amino acid sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 16.

An “aspartate decarboxylase gene” or “ADC gene” as used herein refers toany gene that encodes a polypeptide with aspartate decarboxylaseactivity, meaning the ability to catalyze the conversion of aspartate toβ-alanine. Enzymes having aspartate decarboxylase activity areclassified as EC 4.1.1.11. In certain embodiments, an ADC gene may bederived from a bacterial source. Because an active aspartatedecarboxylase may require proteolytic processing of an inactiveproenzyme, in these embodiments the yeast host cell should be selectedto support formation of an active enzyme coded by a bacterial ADC gene.

In some embodiments, the ADC gene may be derived from an S. avermitilispanD gene encoding the amino acid sequence set forth in SEQ ID NO: 17.In some embodiments, the ADC gene may encode an amino acid sequence withat least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the amino acid sequence of SEQ ID NO: 17. In certain embodiments, anS. avermitilis-derived ADC gene may comprise the nucleotide sequence setforth in any one of SEQ ID NOs: 130, 145, 146, or 147; or a nucleotidesequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the nucleotide sequence set forth in any one of SEQID NOs: 130, 145, 146, or 147.

In other embodiments, the ADC gene may be derived from a C.acetobutylicum panD gene encoding the amino acid sequence set forth inSEQ ID NO: 18. In some embodiments, the ADC gene may encode an aminoacid sequence with at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least99% sequence identity to the amino acid sequence of SEQ ID NO: 18. Incertain embodiments, a C. acetobutylicum-derived ADC gene may comprisethe nucleotide sequence set forth in SEQ ID NO: 131, or a nucleotidesequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the nucleotide sequence set forth in SEQ ID NO:131.

In other embodiments, the ADC gene may be derived from a H. pylori ADCgene encoding the amino acid sequence set forth in SEQ ID NO: 133. Insome embodiments, the ADC gene may encode an amino acid sequence with atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the amino acid sequence of SEQ ID NO: 133. In certain embodiments, aH. pylori-derived ADC gene may comprise the nucleotide sequence setforth in SEQ ID NO: 133, or a nucleotide sequence with at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 133.

In other embodiments, the ADC gene may be derived from a Bacillus sp.TS25 ADC gene encoding the amino acid sequence set forth in SEQ ID NO:135. In some embodiments, the ADC gene may encode an amino acid sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 135. In certainembodiments, a Bacillus sp. TS25-derived ADC gene may comprise thenucleotide sequence set forth in SEQ ID NO: 134, or a nudeotide sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 134.

In other embodiments, the ADC gene may be derived from a C. glutamicumADC gene encoding the amino acid sequence set forth in SEQ ID NO: 137.In some embodiments, the ADC gene may encode an amino acid sequence withat least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the amino acid sequence of SEQ ID NO: 137. In certain embodiments, aC. glutamicum-derived ADC gene may comprise the nucleotide sequence setforth in SEQ ID NO: 136, or a nucleotide sequence with at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 136.

In other embodiments, the ADC gene may be derived from a B.licheniformis ADC gene encoding the amino acid sequence set forth in SEQID NO: 139. In some embodiments, the ADC gene may encode an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 139. Incertain embodiments, a B. licheniformis-derived ADC gene may comprisethe nucleotide sequence set forth in any one of SEQ ID NOs: 138, 148,149, 150, or 151; or a nucleotide sequence with at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99% sequence identity to the nucleotidesequence set forth in any one of SEQ ID NOs: 138, 148, 149, 150, or 151.

A “β-alanine aminotransferase gene” or “BAAT gene” as used herein refersto any gene that encodes a polypeptide with β-alanine aminotransferaseactivity, meaning the ability to catalyze the conversion of β-alanine tomalonate semialdehyde. Enzymes having β-alanine aminotransferaseactivity are classified as EC 2.6.1.19. In certain embodiments, a BAATgene may be derived from a yeast source. For example, a BAAT gene may bederived from the I. orientalis homolog to the pyd4 gene encoding theamino acid sequence set forth in SEQ ID NO: 20. In some embodiments, theBAAT gene may encode an amino acid sequence with at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99% sequence identity to the amino acidsequence of SEQ ID NO: 20. In certain embodiments, an I.orientalis-derived BAAT gene may comprise the nucleotide sequence setforth in SEQ ID NO: 19 or a nucleotide sequence with at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 19. In other embodiments,the BAAT gene may be derived from the S. kluyveni pyd4 gene encoding theamino acid sequence set forth in SEQ ID NO: 21. In some embodiments, theBAAT gene may encode an amino acid sequence with at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99% sequence identity to the amino acidsequence of SEQ ID NO: 21. In certain embodiments, a S. kluyveri-derivedBAAT gene may comprise the nucleotide sequence set forth in SEQ ID NO:142 or a nucleotide sequence with at least 50%, at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, at least97%, or at least 99% sequence identity to the nucleotide sequence setforth in SEQ ID NO: 142. In other embodiments, the BAAT gene may bederived from a bacterial source. For example, a BAAT gene may be derivedfrom an S. avermitilis BAAT gene encoding the amino acid sequence setforth in SEQ ID NO: 22. In some embodiments, the BAAT gene may encode anamino acid sequence with at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to the amino acid sequence of SEQ ID NO: 22.In certain embodiments, a S. avermitilis-derived BAAT gene may comprisethe nucleotide sequence set forth in SEQ ID NO: 140 or a nucleotidesequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the nucleotide sequence set forth in SEQ ID NO:140.

A BAAT gene may also be a “4-aminobutyrate aminotransferase” or “gabTgene” meaning that it has native activity on 4-aminobutyrate as well asβ-alanine. Alternatively, a BAAT gene may be derived by random ordirected engineering of a native gabT gene from a bacterial or yeastsource to encode a polypeptide with BAAT activity. For example, a BAATgene may be derived from the S. avermitilis gabT encoding the amino acidsequence set forth in SEQ ID NO: 23. In some embodiments, the S.avermitilis-derived BAAT gene may encode an amino acid sequence with atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the amino acid sequence of SEQ ID NO: 23. In other embodiments, aBAAT gene may be derived from the S. cerevisiae gabT gene UGA1 encodingthe amino acid sequence set forth in SEQ ID NO: 24. In some embodiments,the S. cerevisiae-derived BAAT gene may encode an amino acid sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 24. In certainembodiments, an S. cerevisiae-derived BAAT gene may comprise thenucleotide sequence set forth in SEQ ID NO: 141 or a nucleotide sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 141.

A “3-HP dehydrogenase gene” or “3-HPDH gene” as used herein refers toany gene that encodes a polypeptide with 3-HP dehydrogenase activity,meaning the ability to catalyze the conversion of malonate semialdehydeto 3-HP. Enzymes having 3-HP dehydrogenase activity are classified as EC1.1.1.59 if they utilize an NAD(H) cofactor, and as EC 1.1.1.298 if theyutilize an NADP(H) cofactor. Enzymes classified as EC 1.1.1.298 arealternatively referred to as malonate semialdehyde reductases.

In certain embodiments, a 3-HPDH gene may be derived from a yeastsource. For example, a 3-HPDH gene may be derived from the I. orientalishomolog to the YMR226C gene encoding the amino acid sequence set forthin SEQ ID NO: 26. In some embodiments, the 3-HPDH gene may encode anamino acid sequence with at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to the amino acid sequence of SEQ ID NO: 26.In certain embodiments, an I. orientalis-derived 3-HPDH gene maycomprise the nucleotide sequence set forth in SEQ ID NO: 25 or anucleotide sequence with at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to the nucleotide sequence set forth in SEQID NO: 25. In other embodiments, a 3-HPDH gene may be derived from theS. cerevisiae YMR226C gene encoding the amino acid sequence set forth inSEQ ID NO: 129. In some embodiments, the 3-HPDH gene may encode an aminoacid sequence with at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least99% sequence identity to the amino acid sequence of SEQ ID NO: 129. Incertain embodiments, an S. cerevisiae-derived 3-HPDH gene may comprisethe nucleotide sequence set forth in SEQ ID NO: 144 or a nucleotidesequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the nucleotide sequence set forth in SEQ ID NO:144.

In other embodiments, the 3-HPDH gene may be derived from a bacterialsource. For example, a 3-HPDH gene may be derived from an E. coli ydfGgene encoding the amino acid sequence in SEQ ID NO: 27. In someembodiments, the gene may encode an amino acid sequence with at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO: 27. In certain embodiments, an E.coli-derived 3-HPDH gene may comprise the nucleotide sequence set forthin SEQ ID NO: 143 or a nucleotide sequence with at least 50%, at least60%, at least 70%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 143. In other embodiments, a 3-HPDHgene may be derived from an M. sedula malonate semialdehyde reductasegene encoding the amino acid sequence set forth in SEQ ID NO: 29. Insome embodiments, the 3-HPDH gene may encode an amino acid sequence withat least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% sequence identityto the amino acid sequence set forth in SEQ ID NO: 29. In certainembodiments, an M. sedula-derived 3-HPDH gene may comprise thenucleotide sequence set forth in SEQ ID NO: 343 or a nucleotide sequencewith at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 343.

A “3-hydroxyisobutyrate dehydrogenase gene” or “HIBADH gene” as usedherein refers to any gene that encodes a polypeptide with3-hydroxyisobutyrate dehydrogenase activity, meaning the ability tocatalyze the conversion of 3-hydroxyisobutyrate to methylmalonatesemialdehyde. Enzymes having 3-hydroxyisobutyrate dehydrogenase activityare classified as EC 1.1.1.31. Some 3-hydroxyisobutyrate dehydrogenasesalso have 3-HPDH activity. In certain embodiments, an HIBADH gene may bederived from a bacterial source. For example, an HIBADH gene may bederived from an A. faecalis M3A gene encoding the amino acid sequenceset forth in SEQ ID NO: 28, a P. putida KT2440 or E23440 mmsB geneencoding the amino acid sequence set forth in SEQ ID NO: 30 or SEQ IDNO: 31, respectively, or a P. aeruginosa PAO1 mmsB gene encoding theamino acid sequence set forth in SEQ ID NO: 32. In certain embodiments,an HIBADH gene may encode an amino acid sequence with at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% sequence identity to the aminoacid sequence set forth in SEQ ID NOs: 28, 30, 31, or 32.

A “4-hydroxybutyrate dehydrogenase gene” as used herein refers to anygene that encodes a polypeptide with 4-hydroxybutyrate dehydrogenaseactivity, meaning the ability to catalyze the conversion of4-hydroxybutanoate to succinate semialdehyde. Enzymes having4-hydroxybutyrate dehydrogenase activity are classified as EC 1.1.1.61.Some 4-hydroxybutyrate dehydrogenases also have 3-HPDH activity. Incertain embodiments, a 4-hydroxybutyrate dehydrogenase gene may bederived from a bacterial source. For example, a 4-hydroxybutyratedehydrogenase gene may be derived from a R. eutropha H16 4hbd geneencoding the amino acid sequence set forth in SEQ ID NO: 33 or a C.kluyveri DSM 555 hbd gene encoding the amino acid sequence set forth inSEQ ID NO: 34. In other embodiments, the gene may encode an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, or at least 99%sequence identity to the amino acid sequence set forth in SEQ ID NOs: 33or 34.

A “PEP carboxykinase gene” or “PCK gene” as used herein refers to anygene that encodes a polypeptide with PEP carboxykinase activity, meaningthe ability to catalyze the conversion of PEP, CO₂, and ADP or GDP toOAA and ATP or GTP, or vice versa. Enzymes having PEP carboxykinaseactivity are classified as EC 4.1.1.32 (GTP/GDP utilizing) and EC4.1.1.49 (ATP/ADP utilizing). In certain embodiments, a PCK gene may bederived from a yeast source. In other embodiments, a PCK gene may bederived from a bacterial source, and in certain of these embodiments thegene may be derived from a bacteria in which the PCK reaction favors theproduction of OAA rather than the more common form of the reaction wheredecarboxylation is dominant. For example, a PCK gene may be derived froman M. succiniciproducens PCK gene encoding the amino acid sequence setforth in SEQ ID NO: 35, an A. succiniciproducens PCK gene encoding theamino acid sequence set forth in SEQ ID NO: 36, an A. succinogenes PCKgene encoding the amino acid sequence set forth in SEQ ID NO: 37, or anR. eutropha PCK gene encoding the amino acid sequence set forth in SEQID NO: 38. In other embodiments, a PCK gene has undergone one or moremutations versus the native gene from which it was derived, such thatthe resultant gene encodes a polypeptide that preferably catalyzes theconversion of PEP to OAA. For example, a PCK gene may be derived from anE. coli K12 strain PCK gene encoding the amino acid sequence set forthin SEQ ID NO: 39, where the gene has been mutated to preferably catalyzethe conversion of PEP to OAA. In other embodiments the conversion of PEPto OAA is catalyzed by a PEP carboxytransphosphorylase such as is foundin propionic acid bacteria (e.g., P. shermanii, A. woodi) which useinorganic phosphate and diphosphate rather than ATP/ADP or GTP/GDP.

A “malate dehydrogenase gene” as used herein refers to any gene thatencodes a polypeptide with malate dehydrogenase activity, meaning theability to catalyze the conversion of OAA to malate. In certainembodiments, a malate dehydrogenase gene may be derived from a bacterialor yeast source.

A “malate decarboxylase gene” as used herein refers to any gene thatencodes a polypeptide with malate decarboxylase activity, meaning theability to catalyze the conversion of malate to 3-HP. Malatedecarboxylase activity is not known to occur naturally. Therefore, amalate decarboxylase gene may be derived by incorporating one or moremutations into a native source gene that encodes a polypeptide withacetolactate decarboxylase activity. Polypeptides with acetolactatedecarboxylase activity catalyze the conversion of2-hydroxy-2-methyl-3-oxobutanoate to 2-acetoin, and are classified as EC4.1.1.5. In certain embodiments, a malate decarboxylase gene may bederived from a bacterial source. For example, a malate decarboxylasegene may be derived from an L. lactis aldB gene encoding the amino acidsequence set forth in SEQ ID NO: 40, an S. thermophilus aldB geneencoding the amino acid sequence set forth in SEQ ID NO: 41, a B. brevisaldB gene encoding the amino acid sequence set forth in SEQ ID NO: 42,or a E. aerogenes budA gene encoding the amino acid sequence set forthin SEQ ID NO: 43.

An “alpha-ketoglutarate (AKG) decarboxylase gene” or “KGD gene” as usedherein refers to any gene that encodes a polypeptide withalpha-ketoglutarate decarboxylase activity, meaning the ability tocatalyze the conversion of alpha-ketoglutarate (2-oxoglutarate) tosuccinate semialdehyde. Enzymes having AKG decarboxylase activity areclassified as EC 4.1.1.71. A KGD gene may be used to derive a geneencoding a polypeptide capable of catalyzing the conversion of OAA tomalonate semialdehyde. This activity may be found in a native KGD gene,or it may derived by incorporating one or more mutations into a nativeKGD gene. In certain embodiments, a KGD gene may be derived from abacterial source. For example, a KGD gene may be derived from a M.tuberculosis KGD gene encoding the amino acid sequence set forth in SEQID NO: 44, a B. japonicum KGD gene encoding the amino acid sequence setforth in SEQ ID NO: 45, or a M. loti (aka Rhizobium loti) KGD geneencoding the amino acid sequence set forth in SEQ ID NO: 46.

A “branched-chain alpha-keto acid decarboxylase gene” or “BCKA gene” asused herein refers to any gene that encodes a polypeptide withbranched-chain alpha-keto acid decarboxylase activity, which can serveto decarboxylate a range of alpha-keto acids from three to six carbonsin length. Enzymes having BCKA activity are classified as EC 4.1.1.72. ABCKA gene may be used to derive a gene encoding a polypeptide capable ofcatalyzing the conversion of OAA to malonate semialdehyde. This activitymay be found in a native BCKA gene, or it may be derived byincorporating one or more mutations into a native BCKA gene. In certainembodiments, a BCKA gene may be derived from a bacterial source. Forexample, a BCKA gene may be derived from a L. lactis kdcA gene encodingthe amino acid sequence set forth in SEQ ID NO: 47.

An “indolepyruvate decarboxylase gene” or “IPDA gene” as used hereinrefers to any gene that encodes a polypeptide with indolepyruvatedecarboxylase activity, meaning the ability to catalyze the conversionof indolepyruvate to indoleacetaldehyde. Enzymes having IPDA activityare classified as EC 4.1.1.74. An IPDA gene may be used to derive a geneencoding a polypeptide capable of catalyzing the conversion of OAA tomalonate semialdehyde. This activity may be found in a native IPDA gene,or it may be derived by incorporating one or more mutations into anative IPDA gene. In certain embodiments, an indolepyruvatedecarboxylase gene may be derived from a yeast, bacterial, or plantsource.

A “pyruvate decarboxylase gene” or “PDC gene” as used herein refers toany gene that encodes a polypeptide with pyruvate decarboxylaseactivity, meaning the ability to catalyze the conversion of pyruvate toacetaldehyde. Enzymes having PDC activity are classified as EC 4.1.1.1.In preferred embodiments, a PDC gene that is incorporated into amodified yeast cell as provided herein has undergone one or moremutations versus the native gene from which it was derived such that theresultant gene encodes a polypeptide capable of catalyzing theconversion of OAA to malonate semialdehyde. In certain embodiments, aPDC gene may be derived from a yeast source. For example, a PDC gene maybe derived from an I. orientalis PDC gene encoding the amino acidsequence set forth in SEQ ID NO: 49, an S. cerevisiae PDC1 gene encodingthe amino acid sequence set forth in SEQ ID NO: 50, or a K. lactis PDCencoding the amino acid sequence set forth in SEQ ID NO: 51. In certainembodiments, a PDC gene derived from the I. orientalis PDC gene maycomprise the nucleotide sequence set forth in SEQ ID NO: 48 or anucleotide sequence with at least 50%, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 99% sequence identity to the nucleotide sequence set forth in SEQID NO: 48. In other embodiments, a PDC gene may be derived from abacterial source. For example, a PDC gene may be derived from a Z.mobilis PDC gene encoding the amino acid sequence set forth in SEQ IDNO: 52 or an A. pasteurianus PDC gene encoding the amino acid sequenceset forth in SEQ ID NO: 53.

A “benzoylformate decarboxylase” gene as used herein refers to any genethat encodes a polypeptide with benzoylformate decarboxylase activity,meaning the ability to catalyze the conversion of benzoylformate tobenzaldehyde. Enzymes having benzoylformate decarboxylase activity areclassified as EC 4.1.1.7. In preferred embodiments, a benzoylformatedecarboxylase gene that is incorporated into a modified yeast cell asprovided herein has undergone one or more mutations versus the nativegene from which it was derived such that the resultant gene encodes apolypeptide capable of catalyzing the conversion of OAA to malonatesemialdehyde. In certain embodiments, a benzoylformate decarboxylasegene may be derived from a bacterial source. For example, abenzoylformate decarboxylase gene may be derived from a P. putida mdlCgene encoding the amino acid sequence set forth in SEQ ID NO: 54, a P.aeruginosa mdlC gene encoding the amino acid sequence set forth in SEQID NO: 55, a P. stutzeri dpgB gene encoding the amino acid sequence setforth in SEQ ID NO: 56, or a P. fluorescens ilvB-1 gene encoding theamino acid sequence set forth in SEQ ID NO: 57.

An “OAA formatelyase gene” as used herein refers to any gene thatencodes a polypeptide with OAA formatelyase activity, meaning theability to catalyze the conversion of an acylate ketoacid to itscorresponding CoA derivative. A polypeptide encoded by an OAAformatelyase gene may have activity on pyruvate or on another ketoacid.In certain embodiments, an OAA formatelyase gene encodes a polypeptidethat converts OAA to malonyl-CoA.

A “malonyl-CoA reductase gene” as used herein refers to any gene thatencodes a polypeptide with malonyl-CoA reductase activity, meaning theability to catalyze the conversion of malonyl-CoA to malonatesemialdehyde (also referred to as Co-A acylating malonate semialdehydedehydrogenase activity). In certain embodiments, a malonyl-CoA reductasegene may be derived from a bifunctional malonyl-CoA reductase gene whichalso has the ability to catalyze the conversion of malonate semialdehydeto 3-HP. In certain of these embodiments, a malonyl-CoA reductase genemay be derived from a bacterial source. For example, a malonyl-CoAreductase gene may be derived from a C. aurantiacus malonyl-CoAreductase gene encoding the amino acid sequence set forth in SEQ ID NO:58, an R. castenholzii malonyl-CoA reductase gene encoding the aminoacid sequence set forth in SEQ ID NO: 59, or an Erythrobacter sp. NAP1malonyl-CoA reductase gene encoding the amino acid sequence set forth inSEQ ID NO: 60. In other embodiments, a malonyl-CoA reductase gene may bederived from a malonyl-CoA reductase gene encoding a polypeptide thatonly catalyzes the conversion of malonyl-CoA to malonate semialdehyde.For example, a malonyl-CoA reductase gene may be derived from an M.sedula Msed_0709 gene encoding the amino acid sequence set forth in SEQID NO: 61 or a S. tokodaii malonyl-CoA reductase encoding the amino acidsequence set forth in SEQ ID NO: 62.

A “pyruvate dehydrogenase gene” or “PDH gene” as used herein refers toany gene that encodes a polypeptide with pyruvate dehydrogenaseactivity, meaning the ability to catalyze the conversion of pyruvate toacetyl-CoA. In certain embodiments, a PDH gene may be derived from ayeast source. For example, a PDH gene may be derived from an S.cerevisiae LAT1, PDA1, PDB1, or LPD gene encoding the amino acidsequence set forth in SEQ ID NOs: 63-66, respectively. In otherembodiments, a PDH gene may be derived from a bacterial source. Forexample, a PDH gene may be derived from an E. coli strain K12 substr.MG1655 aceE, aceF, or Ipd gene encoding the amino acid sequence setforth in SEQ ID NOs: 67-69, respectively, or a B. subtilis pdhA, pdhB,pdhC, or pdhD gene encoding the amino acid sequence set forth in SEQ IDNOs: 70-73, respectively.

An “acetyl-CoA carboxylase gene” or “ACC gene” as used herein refers toany gene that encodes a polypeptide with acetyl-CoA carboxylaseactivity, meaning the ability to catalyze the conversion of acetyl-CoAto malonyl-CoA. Enzymes having acetyl-CoA carboxylase activity areclassified as EC 6.4.1.2. In certain embodiments, an acetyl-CoAcarboxylase gene may be derived from a yeast source. For example, anacetyl-CoA carboxylase gene may be derived from an S. cerevisiae ACC1gene encoding the amino acid sequence set forth in SEQ ID NO: 74. Inother embodiments, an acetyl-CoA carboxylase gene may be derived from abacterial source. For example, an acetyl-CoA carboxylase gene may bederived from an E. coli accA, accB, accC, or accD gene encoding theamino acid sequence set forth in SEQ ID NOs: 75-78, respectively, or aC. aurantiacus accA, accB, accC, or accD gene encoding the amino acidsequence set forth in SEQ ID NOs: 79-82, respectively.

An “alanine dehydrogenase gene” as used herein refers to any gene thatencodes a polypeptide with alanine dehydrogenase activity, meaning theability to catalyze the NAD-dependent reductive amination of pyruvate toalanine. Enzymes having alanine dehydrogenase activity are classified asEC 1.4.1.1. In certain embodiments, an alanine dehydrogenase gene may bederived from a bacterial source. For example, an alanine dehydrogenasegene may be derived from an B. subtilis alanine dehydrogenase geneencoding the amino acid sequence set forth in SEQ ID NO: 83.

A “pyruvate/alanine aminotransferase gene” as used herein refers to anygene that encodes a polypeptide with pyruvate/alanine aminotransferaseactivity, meaning the ability to catalyze the conversion of pyruvate andL-glutamate to alanine and 2-oxoglutarate. In certain embodiments, apyruvate/alanine aminotransferase gene is derived from a yeast source.For example, a pyruvate/alanine aminotransferase gene may be derivedfrom an S. pombe pyruvate/alanine aminotransferase gene encoding theamino acid sequence set forth in SEQ ID NO: 84 or an S. cerevisiae ALT2gene encoding the amino acid sequence set forth in SEQ ID NO: 85.

An “alanine 2,3 aminomutase gene” or “AAM gene” as used herein refers toa gene that encodes a polypeptide with alanine 2,3 aminomutase activity,meaning the ability to catalyze the conversion of alanine to β-alanine.Alanine 2,3 aminomutase activity is not known to occur naturally.Therefore, an alanine 2,3 aminomutase gene can be derived byincorporating one or more mutations into a native source gene thatencodes a polypeptide with similar activity such as lysine 2,3aminomutase activity (see, e.g., U.S. Pat. No. 7,309,597). In certainembodiments, the native source gene may be a B. subtilis lysine 2,3aminomutase gene encoding the amino acid sequence set forth in SEQ IDNO: 86, a P. gingivalis lysine 2,3 aminomutase gene encoding the aminoacid sequence set forth in SEQ ID NO: 87, or a F. nucleatum (ATCC-10953)lysine 2,3 aminomutase gene encoding the amino acid sequence set forthin SEQ ID NO: 88.

A “CoA transferase gene” as used herein refers to any gene that encodesa polypeptide with CoA transferase activity, which in one exampleincludes the ability to catalyze the conversion of β-alanine toβ-alanyl-CoA and/or the conversion of lactate to lactyl-CoA. In certainembodiments, a CoA transferase gene may be derived from a yeast source.In other embodiments, a CoA transferase gene may be derived from abacterial source. For example, a CoA transferase gene may be derivedfrom an M. elsdenii CoA transferase gene encoding the amino acidsequence set forth in SEQ ID NO: 89.

A “CoA synthetase gene” as used herein refers to any gene that encodes apolypeptide with CoA synthetase activity. In one example this includesthe ability to catalyze the conversion of β-alanine to β-alanyl-CoA. Inanother example, this includes the ability to catalyze the conversion oflactate to lactyl-CoA. In certain embodiments, a CoA synthetase gene maybe derived from a yeast source. For example, a CoA synthetase gene maybe derived from an S. cerevisiae CoA synthetase gene. In otherembodiments, a CoA synthetase gene may be derived from a bacterialsource. For example, a CoA synthetase gene may be derived from an E.coli CoA synthetase, R. sphaeroides, or S. enterica CoA synthetase gene.

A “β-alanyl-CoA ammonia lyase gene” as used herein refers to any genethat encodes a polypeptide with β-alanyl-CoA ammonia lyase activity,meaning the ability to catalyze the conversion of β-alanyl-CoA toacrylyl-CoA. In certain embodiments, a β-alanyl-CoA ammonia lyase genemay be derived from a bacterial source, such as a C. propionicumβ-alanyl-CoA ammonia lyase gene encoding the amino acid sequence setforth in SEQ ID NO: 90.

A “3-HP-CoA dehydratase gene” or “acrylyl-CoA hydratase gene” as usedherein refers to any gene that encodes a polypeptide with 3-HP-CoAdehydratase gene activity, meaning the ability to catalyze theconversion of acrylyl-CoA to 3-HP-CoA. Enzymes having 3-HP-CoAdehydratase activity are classified as EC 4.2.1.116. In certainembodiments, a 3-HP-CoA dehydratase gene may be derived from a yeast orfungal source, such as a P. sojae 3-HP-CoA dehydratase gene encoding theamino acid sequence set forth in SEQ ID NO: 91. In other embodiments, a3-HP-CoA dehydratase gene may be derived from a bacterial source. Forexample, a 3-HP-CoA dehydratase gene may be derived from a C.aurantiacus 3-HP-CoA dehydratase gene encoding the amino acid sequenceset forth in SEQ ID NO: 92, an R. rubrum 3-HP-CoA dehydratase geneencoding the amino acid sequence set forth in SEQ ID NO: 93, or an R.capsulates 3-HP-CoA dehydratase gene encoding the amino acid sequenceset forth in SEQ ID NO: 94. In still other embodiments, a 3-HP-CoAdehydratase gene may be derived from a mammalian source. For example, a3-HP-CoA dehydratase gene may be derived from a H. sapiens 3-HP-CoAdehydratase gene encoding the amino acid sequence set forth in SEQ IDNO: 95.

A “3-HP-CoA hydrolase gene” as used herein refers to any gene thatencodes a polypeptide with 3-HP-CoA hydrolase activity, meaning theability to catalyze the conversion of 3-HP-CoA to 3-HP. In certainembodiments, a 3-HP-CoA gene may be derived from a yeast or fungalsource. In other embodiments, a 3-HP-CoA gene may be derived from abacterial or mammalian source.

A “3-hydroxyisobutyryl-CoA hydrolase gene” as used herein refers to anygene that encodes a polypeptide with 3-hydroxyisobutyryl-CoA hydrolaseactivity, which in one example includes the ability to catalyze theconversion of 3-HP-CoA to 3-HP. In certain embodiments, a3-hydroxyisobutyryl-CoA hydrolase gene may be derived from a bacterialsource, such as a P. fluorescens 3-hydroxyisobutyryl-CoA hydrolase geneencoding the amino acid sequence set forth in SEQ ID NO: 96 or a B.cereus 3-hydroxyisobutyryl-CoA hydrolase gene encoding the amino acidsequence set forth in SEQ ID NO: 97. In other embodiments, a3-hydroxyisobutyryl-CoA hydrolase gene may be derived from a mammaliansource, such as a H. sapiens 3-hydroxyisobutyryl-CoA hydrolase geneencoding the amino acid sequence set forth in SEQ ID NO: 98.

A “lactate dehydrogenase gene” or “LDH gene” as used herein refers toany gene that encodes a polypeptide with lactate dehydrogenase activity,meaning the ability to catalyze the conversion of pyruvate to lactate.In certain embodiments, an LDH gene may be derived from a fungal,bacterial, or mammalian source.

A “lactyl-CoA dehydratase gene” as used herein refers to any gene thatencodes a polypeptide with lactyl-CoA dehydratase activity, meaning theability to catalyze the conversion of lactyl-CoA to acrylyl-CoA. Incertain embodiments, a lactyl-CoA dehydratase gene may be derived from abacterial source. For example, a lactyl-CoA dehydratase gene may bederived from an M. elsdenii lactyl-CoA dehydratase E1, EIIa, or EIIbsubunit gene encoding the amino acid sequence set forth in SEQ ID NOs:99-101.

An “aldehyde dehydrogenase gene” as used herein refers to any gene thatencodes a polypeptide with aldehyde dehydrogenase activity, which in oneexample includes the ability to catalyze the conversion of 3-HPA to 3-HPand vice versa. In certain embodiments, an aldehyde dehydrogenase genemay be derived from a yeast source, such as an S. cerevisiae aldehydedehydrogenase gene encoding the amino acid sequence set forth in SEQ IDNO: 102 or an I. orientalis aldehyde dehydrogenase gene encoding theamino acid sequence set forth in SEQ ID NOs: 122, 124, or 126. In otherembodiments, an aldehyde dehydrogenase may be derived from a bacterialsource, such as an E. coli aldH gene encoding the amino acid sequenceset forth in SEQ ID NO: 103 or a K. pneumoniae aldehyde dehydrogenasegene encoding the amino acid sequence set forth in SEQ ID NO: 104.

A “glycerol dehydratase gene” as used herein refers to any gene thatencodes a polypeptide with glycerol dehydratase activity, meaning theability to catalyze the conversion of glycerol to 3-HPA. In certainembodiments, a glycerol dehydratase gene may be derived from a bacterialsource, such as a K. pneumonia or C. freundii glycerol dehydratase gene.

In certain embodiments, the genetically modified yeast cells providedherein further comprise a deletion or disruption of one or more nativegenes. “Deletion or disruption” with regard to a native gene means thateither the entire coding region of the gene is eliminated (deletion) orthe coding region of the gene, its promoter, and/or its terminatorregion is modified (such as by deletion, insertion, or mutation) suchthat the gene no longer produces an active enzyme, produces a severelyreduced quantity (at least 75% reduction, preferably at least 90%reduction) of an active enzyme, or produces an enzyme with severelyreduced (at least 75% reduced, preferably at least 90% reduced)activity.

In certain embodiments, deletion or disruption of one or more nativegenes results in a deletion or disruption of one or more nativemetabolic pathways. “Deletion or disruption” with regard to a metabolicpathway means that the pathway is either inoperative or else exhibitsactivity that is reduced by at least 75%, at least 85%, or at least 95%relative to the native pathway. In certain embodiments, deletion ordisruption of a native metabolic pathway is accomplished byincorporating one or more genetic modifications that result in decreasedexpression of one or more native genes that reduce 3-HP production.

In certain embodiments, deletion or disruption of native gene can beaccomplished by forced evolution, mutagenesis, or genetic engineeringmethods, followed by appropriate selection or screening to identify thedesired mutants. In certain embodiments, deletion or disruption of anative host cell gene may be coupled to the incorporation of one or moreexogenous genes into the host cell, i.e., the exogenous genes may beincorporated using a gene expression integration construct that is alsoa deletion construct. In other embodiments, deletion or disruption maybe accomplished using a deletion construct that does not contain anexogenous gene or by other methods known in the art.

In certain embodiments, the genetically modified yeast cells providedherein comprise a deletion or disruption of one or more native genesencoding an enzyme involved in ethanol fermentation, including forexample pyruvate decarboxylase (PDC, converts pyruvate to acetaldehyde)and/or alcohol dehydrogenase (ADH, converts acetaldehyde to ethanol)genes. These modifications decrease the ability of the yeast cell toproduce ethanol, thereby maximizing 3-HP production. However, in certainembodiments the genetically modified yeast cells provided herein may beengineered to co-produce 3-HP and ethanol. In those embodiments, nativegenes encoding an enzyme involved in ethanol fermentation are preferablynot deleted or disrupted, and in certain embodiments the yeast cells maycomprise one or more exogenous genes that increase ethanol production.

In certain embodiments, the genetically modified yeast cells providedherein comprise a deletion or disruption of one or more native genesencoding an enzyme involved in producing alternate fermentative productssuch as glycerol or other byproducts such as acetate or diols. Forexample, the cells provided herein may comprise a deletion or disruptionof one or more of glycerol 3-phosphate dehydrogenase (GPD, catalyzesreaction of dihydroxyacetone phosphate to glycerol 3-phosphate),glycerol 3-phosphatase (GPP, catalyzes conversion of glycerol-3phosphate to glycerol), glycerol kinase (catalyzes conversion ofglycerol 3-phosphate to glycerol), dihydroxyacetone kinase (catalyzesconversion of dihydroxyacetone phosphate to dihydroxyacetone), glyceroldehydrogenase (catalyzes conversion of dihydroxyacetone to glycerol),aldehyde dehydrogenase (ALD, e.g., converts acetaldehyde to acetate or3-HP to 3-HPA), or butanediol dehydrogenase (catalyzes conversion ofbutanediol to acetoin and vice versa) genes.

In certain embodiments, the genetically modified yeast cells providedherein comprise a deletion or disruption of one or more native genesencoding an enzyme that catalyzes a reverse reaction in a 3-HPfermentation pathway, including for example PEP carboxykinase (PCK),enzymes with OAA decarboxylase activity, or CYB2A or CYB2B (catalyzesthe conversion of lactate to pyruvate). PCK catalyzes the conversion ofPEP to OAA and vice versa, but exhibits a preference for the OAA to PEPreaction. To reduce the conversion of OAA to PEP, one or more copies ofa native PCK gene may be deleted or disrupted. In certain embodiments,yeast cells in which one or more native PCK genes have been deleted ordisrupted may express one or more exogenous PCK genes that have beenmutated to encode a polypeptide that favors the conversion of PEP toOAA. OAA decarboxylase catalyzes the conversion of OAA to pyruvate.Enzymes with OAA decarboxylase activity have been identified, such asthat coded by the eda gene in E. coli and malic enzyme (MAE) in yeastand fungi. To reduce OAA decarboxylase activity, one or more copies of anative gene encoding an enzyme with OAA decarboxylase activity may bedeleted or disrupted. In certain embodiments, yeast cells in which oneor more native OAA decarboxylation genes have been deleted or disruptedmay express one or more exogenous OAA decarboxylation genes that havebeen mutated to encode a polypeptide that catalyzes the conversion ofpyruvate to OAA.

In certain embodiments, the genetically modified yeast cells providedherein comprise a deletion or disruption of one or more native genesencoding an enzyme involved in an undesirable reaction with a 3-HPfermentation pathway product or intermediate. Examples of such genesinclude those encoding an enzyme that converts 3HP to an aldehyde of3HP, which are known to be toxic to bacterial cells.

In certain embodiments, the genetically modified yeast cells providedherein comprise a deletion or disruption of one or more native genesencoding an enzyme that has a neutral effect on a 3-HP fermentationpathway, including for example GAL6 (negative regulator of the GALsystem that converts galactose to glucose). Deletion or disruption ofneutral genes allows for insertion of one or more exogenous geneswithout affecting native fermentation pathways.

In certain embodiments, the yeast cells provided herein are 3-HPresistant yeast cells. A “3-HP-resistant yeast cell” as used hereinrefers to a yeast cell that exhibits an average glycolytic rate of atleast 2.5 g/L/hr in media containing 75 g/L or greater 3-HP at a pH ofless than 4.0. Such rates and conditions represent an economic processfor producing 3-HP. In certain of these embodiments, the yeast cells mayexhibit 3-HP resistance in their native form. In other embodiments, thecells may have undergone mutation and/or selection (e.g., chemostatselection or repeated serial subculturing) before, during, or afterintroduction of genetic modifications related to an active 3-HPfermentation pathway, such that the mutated and/or selected cellspossess a higher degree of resistant to 3-HP than wild-type cells of thesame species. For example, in some embodiments, the cells have undergonemutation and/or selection in the presence of 3-HP or lactic acid beforebeing genetically modified with one or more exogenous 3-HP pathwaygenes. In certain embodiments, mutation and/or selection may be carriedout on cells that exhibit 3-HP resistance in their native form. Cellsthat have undergone mutation and/or selection may be tested for sugarconsumption and other characteristics in the presence of varying levelsof 3-HP in order to determine their potential as industrial hosts for3-HP production. In addition to 3-HP resistance, the yeast cellsprovided herein may have undergone mutation and/or selection forresistance to one or more additional organic acids (e.g., lactic acid)or to other fermentation products, byproducts, or media components.

Selection, such as selection for resistance to 3-HP or to othercompounds, may be accomplished using methods well known in the art. Forexample, as mentioned supra, selection may be chemostat selection.Chemostat selection uses a chemostat that allows for a continuousculture of microorganisms (e.g., yeast) wherein the specific growth rateand cell number can be controlled independently. A continuous culture isessentially a flow system of constant volume to which medium is addedcontinuously and from which continuous removal of any overflow canoccur. Once such a system is in equilibrium, cell number and nutrientstatus remain constant, and the system is in a steady state. A chemostatallows control of both the population density and the specific growthrate of a culture through dilution rate and alteration of theconcentration of a limiting nutrient, such as a carbon or nitrogensource. By altering the conditions as a culture is grown (e.g.,decreasing the concentration of a secondary carbon source necessary tothe growth of the inoculum strain, among others), microorganisms in thepopulation that are capable of growing faster at the altered conditionswill be selected and will outgrow microorganisms that do not function aswell under the new conditions. Typically such selection requires theprogressive increase or decrease of at least one culture component overthe course of growth of the chemostat culture. The operation ofchemostats and their use in the directed evolution of microorganisms iswell known in the art (see, e.g., Novick Proc Natl Acad Sci USA36:708-719 (1950), Harder J Appl Bacteriol 43:1-24 (1977). Other methodsfor selection include, but are not limited to, repeated serialsubculturing under the selective conditions as described in e.g., U.S.Pat. No. 7,629,162. Such methods can be used in place of, or in additionto, using the glucose limited chemostat method described above.

Yeast strains exhibiting the best combinations of growth and glucoseconsumption in 3-HP media as disclosed in the examples below arepreferred host cells for various genetic modifications relating to 3-HPfermentation pathways. Yeast genera that possess the potential for arelatively high degree of 3-HP resistance, as indicated by growth in thepresence of 75 g/L 3-HP or higher at a pH of less than 4, include forexample Candida, Kluyveromyces, Issatchenkia, Saccharomyces, Pichia,Schizosaccharomyces, Torulaspora, and Zygosaccharomyces. Speciesexhibiting 3-HP resistance included I. orientalis (also known as C.krusei), C. lambica (also known as Pichia fermentans), and S. bulderi(also known as Kazachstania bulderi). I. orientalis and C. lambica arefrom the I. orientalis/P. fermentans clade, while S. bulderi is from theSaccharomyces clade. Specific strains exhibiting 3-HP resistanceincluded I. orientalis strains 24210, PTA-6658, 60585, and CD1822, S.bulderi strains MYA-402 and MYA-404, and C. lambica strain ATCC 38617.

Other wild-type yeast or fungi may be tested in a similar manner andidentified to have acceptable levels of growth and glucose utilizationin the presence of high levels of 3-HP as described herein. For example,Gross and Robbins (Hydrobiologia 433(103):91-109) have compiled a listof 81 fungal species identified in low pH (<4) environments that couldbe relevant to test as potential production hosts.

In certain embodiments, the modified yeast cells provided herein aregenerated by incorporating one or more genetic modifications into aCrabtree-negative host yeast cell. In certain of these embodiments thehost yeast cell belongs to the genus Issatchenkia, Candida, orSaccharomyces, and in certain of these embodiments the host cell belongsto the I. orientalis/P. fermentans or Saccharomyces clade. In certain ofembodiments, the host cell is I. orientalis or C. lambica, or S.bulderi.

The I. orientalis/P. fermentans clade is the most terminal clade thatcontains at least the species I. orientalis, P. galeiformis, P. sp.YB-4149 (NRRL designation), C. ethanolica, P. deserticola, P.membranifaciens, and P. fermentans. Members of the I. orientalis/P.fermentans clade are identified by analysis of the variable D1/D2 domainof the 26S ribosomal DNA of yeast species, using the method described byKurtzman and Robnett in “Identification and Phylogeny of AscomycetousYeasts from Analysis of Nuclear Large Subunit (26S) Ribosomal DNAPartial Sequences,” Antonie van Leeuwenhoek 73:331-371, 1998,incorporated herein by reference (see especially p. 349). Analysis ofthe variable D1/D2 domain of the 26S ribosomal DNA from hundreds ofascomycetes has revealed that the I. orientalis/P. fermentans cladecontains very closely related species. Members of the I. orientalis/P.fermentans clade exhibit greater similarity in the variable D1/D2 domainof the 26S ribosomal DNA to other members of the clade than to yeastspecies outside of the clade. Therefore, other members of the I.orientalis/P. fermentans clade can be identified by comparison of theD1/D2 domains of their respective ribosomal DNA and comparing to that ofother members of the clade and closely related species outside of theclade, using Kurtzman and Robnett's methods.

In certain embodiments, the genetically modified yeast cells providedherein belong to the genus Issatchenkia, and in certain of theseembodiments the yeast cells are I. orientalis. When first characterized,the species I. orientalis was assigned the name Pichia kudriavzevii. Theanamorph (asexual form) of I. orientalis is known as Candida krusei.Numerous additional synonyms for the species I. orientalis have beenlisted elsewhere (Kurtzman and Fell, The Yeasts, a Taxonomic Study.Section 35. Issatchenkia Kudryavtsev, pp 222-223 (1998)).

The ideal yeast cell for 3-HP production is ideally capable of growingat low pH levels. The ability to conduct fermentation at a low pHdecreases downstream recovery costs, resulting in more economicalproduction. Therefore, in certain embodiments the yeast host cell iscapable of growing at low pH levels (e.g., at pH levels less than 7, 6,5, 4, or 3).

A suitable host cell may possess one or more favorable characteristicsin addition to 3-HP resistance and/or low pH growth capability. Forexample, potential host cells exhibiting 3-HP resistance may be furtherselected based on glycolytic rates, specific growth rates,thermotolerance, tolerance to biomass hydrolysate inhibitors, overallprocess robustness, and so on. These criteria may be evaluated prior toany genetic modification relating to a 3-HP fermentation pathway, orthey may be evaluated after one or more such modifications have takenplace.

Because most yeast are native producers of ethanol, elimination orsevere reduction in the enzyme catalyzing the first step in ethanolproduction from pyruvate (PDC) is required for sufficient yield of analternate product. In Crabtree-positive yeast such as Saccharomyces, adeleted or disrupted PDC gene causes the host to acquire an auxotrophyfor two-carbon compounds such as ethanol or acetate, and causes a lackof growth in media containing glucose. Mutants capable of overcomingthese limitations can be obtained using progressive selection foracetate independence and glucose tolerance (see, e.g., van Maris ApplEnviron Microbiol 70:159 (2004)). Therefore, in certain embodiments apreferred yeast host cell is a Crabtree-negative yeast cell, in whichPDC deletion strains are able to grow on glucose and retain C2prototrophy.

The level of gene expression and/or the number of exogenous genes to beutilized in a given cell will vary depending on the yeast speciesselected. For fully genome-sequenced yeasts, whole-genome stoichiometricmodels may be used to determine which enzymes should be expressed todevelop a desired pathway 3-HP fermentation pathway. Whole-genomestoichiometric models are described in, for example, Hjersted et al.,“Genome-scale analysis of Saccharomyces cerevisiae metabolism andethanol production in fed-batch culture,” Biotechnol. Bioeng. 2007; andFamili et al., “Saccharomyces cerevisiae phenotypes can be predicted byusing constraint-based analysis of a genome-scale reconstructedmetabolic network,” Proc. Natl. Acad. Sci. 2003, 100(23):13134-9.

For yeasts without a known genome sequence, sequences for genes ofinterest (either as overexpression candidates or as insertion sites) cantypically be obtained using techniques such as those described below inExample 2A. Routine experimental design can be employed to testexpression of various genes and activity of various enzymes, includinggenes and enzymes that function in a 3-HP pathway. Experiments may beconducted wherein each enzyme is expressed in the yeast individually andin blocks of enzymes up to and including preferably all pathway enzymes,to establish which are needed (or desired) for improved 3-HP production.One illustrative experimental design tests expression of each individualenzyme as well as of each unique pair of enzymes, and further can testexpression of all required enzymes, or each unique combination ofenzymes. A number of approaches can be taken, as will be appreciated.

In certain embodiments, methods are provided for producing 3-HP from agenetically modified yeast cell as provided herein. In certainembodiments, these methods comprise culturing a genetically modifiedyeast cell as provided herein in the presence of at least one carbonsource, allowing the cell to produce 3-HP for a period of time, and thenisolating 3-HP produced by the cell from culture. The carbon source maybe any carbon source that can be fermented by the provided yeast. Thecarbon source may be a twelve carbon sugar such as sucrose, a hexosesugar such as glucose or fructose, glycan or other polymer of glucose,glucose oligomers such as maltose, maltotriose and isomaltotriose,panose, and fructose oligomers. If the cell is modified to impart anability to ferment pentose sugars, the fermentation medium may include apentose sugar such as xylose, xylan or other oligomer of xylose, and/orarabinose. Such pentose sugars are suitably hydrolysates of ahemicellulose-containing biomass. In the case of oligomeric sugars, itmay be necessary to add enzymes to the fermentation broth in order todigest these to the corresponding monomeric sugar for fermentation bythe cell. In certain embodiments, more than one type of geneticallymodified yeast cell may be present in the culture. Likewise, in certainembodiments one or more native yeast cells of the same or a differentspecies than the genetically modified yeast cell may be present in theculture.

In certain embodiments, culturing of the cells provided herein toproduce 3-HP may be divided up into phases. For example, the cellculture process may be divided into a cultivation phase, a productionphase, and a recovery phase. One of ordinary skill in the art willrecognize that the conditions used for these phases may be varied basedon factors such as the species of yeast being used, the specific 3-HPfermentation pathway utilized by the yeast, the desired yield, or otherfactors.

The medium will typically contain nutrients as required by theparticular cell, including a source of nitrogen (such as amino acids,proteins, inorganic nitrogen sources such as ammonia or ammonium salts,and the like), and various vitamins, minerals and the like. In someembodiments, the cells of the invention can be cultured in a chemicallydefined medium. In one example, the medium contains around 5 g/Lammonium sulfate, around 3 g/L potassium dihydrogen phosphate, around0.5 g/L magnesium sulfate, trace elements, vitamins and around 150 g/Lglucose. The pH may be allowed to range freely during cultivation, ormay be buffered if necessary to prevent the pH from falling below orrising above predetermined levels. In certain embodiments, thefermentation medium is inoculated with sufficient yeast cells that arethe subject of the evaluation to produce an OD₆₀₀ of about 1.0. Unlessexplicitly noted otherwise, OD₆₀₀ as used herein refers to an opticaldensity measured at a wavelength of 600 nm with a 1 cm pathlength usinga model DU600 spectrophotometer (Beckman Coulter). The cultivationtemperature may range from around 30-40° C., and the cultivation timemay be up to around 120 hours.

In one example, the concentration of cells in the fermentation medium istypically in the range of about 0.1 to 20, preferably from 0.1 to 5,even more preferably from 1 to 3 g dry cells/liter of fermentationmedium during the production phase. The fermentation may be conductedaerobically, microaerobically, or anaerobically, depending on pathwayrequirements. If desired, oxygen uptake rate (OUR) can be variedthroughout fermentation as a process control (see, e.g., WO03/102200).In some embodiments, the modified yeast cells provided herein arecultivated under microaerobic conditions characterized by an oxygenuptake rate from 2 to 45 mmol/L/hr, e.g., 2 to 25, 2 to 20, 2 to 15, 2to 10, 10 to 45, 15 to 40, 20 to 35, or 25 to 35 mmol/L/hr. In certainembodiments, the modified yeast cells provided herein may performespecially well when cultivated under microaerobic conditionscharacterized by an oxygen uptake rate of from 2 to 25 mmol/L/hr. Themedium may be buffered during the production phase such that the pH ismaintained in a range of about 3.0 to about 7.0, or from about 4.0 toabout 6.0. Suitable buffering agents are basic materials that neutralizethe acid as it is formed, and include, for example, calcium hydroxide,calcium carbonate, sodium hydroxide, potassium hydroxide, potassiumcarbonate, sodium carbonate, ammonium carbonate, ammonia, ammoniumhydroxide and the like. In general, those buffering agents that havebeen used in conventional fermentation processes are also suitable here.

In those embodiments where a buffered fermentation is utilized, acidicfermentation products may be neutralized to the corresponding salt asthey are formed. In these embodiments, recovery of the acid involvesregeneration of the free acid. This may be done by removing the cellsand acidulating the fermentation broth with a strong acid such assulfuric acid. This results in the formation of a salt by-product. Forexample, where a calcium salt is utilized as the neutralizing agent andsulfuric acid is utilized as the acidulating agent, gypsum is producedas a salt by-product. This by-product is separated from the broth, andthe acid is recovered using techniques such as liquid-liquid extraction,distillation, absorption, and others (see, e.g., T. B. Vickroy, Vol. 3,Chapter 38 of Comprehensive Biotechnology, (ed. M. Moo-Young), Pergamon,Oxford, 1985; R. Datta, et al., FEMS Microbiol Rev, 1995, 16:221-231;U.S. Pat. Nos. 4,275,234, 4,771,001, 5,132,456, 5,420,304, 5,510,526,5,641,406, and 5,831,122, and WO93/00440.

In other embodiments, the pH of the fermentation medium may be permittedto drop during cultivation from a starting pH that is at or above thepKa of 3-HP, typically 4.5 or higher, to at or below the pKa of the acidfermentation product, e.g., less than 4.5 or 4.0, such as in the rangeof about 1.5 to about 4.5, in the range of from about 2.0 to about 4.0,or in the range from about 2.0 to about 3.5.

In still other embodiments, fermentation may be carried out to produce aproduct acid by adjusting the pH of the fermentation broth to at orbelow the pKa of the product acid prior to or at the start of thefermentation process. The pH may thereafter be maintained at or belowthe pKa of the product acid throughout the cultivation. In certainembodiments, the pH may be maintained at less than 4.5 or 4.0, such asin a range of about 1.5 to about 4.5, in a range of about 2.0 to about4.0, or in a range of about 2.0 to about 3.5.

In certain embodiments of the methods provided herein, the geneticallymodified yeast cells produce relatively low levels of ethanol. Incertain embodiments, ethanol may be produced in a yield of 10% or less,preferably in a yield of 2% or less. In certain of these embodiments,ethanol is not detectably produced. In other embodiments, however, 3-HPand ethanol may be co-produced. In these embodiments, ethanol may beproduced at a yield of greater than 10%, greater than 25%, or greaterthan 50%.

In certain embodiments of the methods provided herein, the final yieldof 3-HP on the carbon source is at least 10%, at least 20%, at least30%, at least 40%, at least 50%, or greater than 50% of the theoreticalyield. The concentration, or titer, of 3-HP will be a function of theyield as well as the starting concentration of the carbon source. Incertain embodiments, the titer may reach at least 1-3, at least 5, atleast 10, at least 20, at least 30, at least 40, at least 50, or greaterthan 50 g/L at some point during the fermentation, and preferably at theend of the fermentation. In certain embodiments, the final yield of 3-HPmay be increased by altering the temperature of the fermentation medium,particularly during the production phase.

Once produced, any method known in the art can be used to isolate 3-HPfrom the fermentation medium. For example, common separation techniquescan be used to remove the biomass from the broth, and common isolationprocedures (e.g., extraction, distillation, and ion-exchange procedures)can be used to obtain the 3-HP from the microorganism-free broth. Inaddition, 3-HP can be isolated while it is being produced, or it can beisolated from the broth after the product production phase has beenterminated.

3-HP produced using the methods disclosed herein can be chemicallyconverted into other organic compounds. For example, 3-HP can behydrogenated to form 1,3 propanediol, a valuable polyester monomer.Propanediol also can be created from 3-HP using polypeptides havingoxidoreductase activity in vitro or in vivo. Hydrogenating an organicacid such as 3-HP can be performed using any method such as those usedto hydrogenate succinic acid and/or lactic acid. For example, 3-HP canbe hydrogenated using a metal catalyst. In another example, 3-HP can bedehydrated to form acrylic acid using any known method for performingdehydration reactions. For example, 3-HP can be heated in the presenceof a catalyst (e.g., a metal or mineral acid catalyst) to form acrylicacid.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Media and Solutions

TE was composed of 10 mM Tris Base and 1 mM EDTA, pH 8.0.2× YT+amp plates were composed of 16 g/L tryptone, 10 g/L yeast extract,5 g/L NaCl, 100 mg/L ampicillin, and 15 g/L Bacto agar.ura selection plates were composed of 6.7 g yeast nitrogen base withammonium sulfate, 5 g casamino acids, 100 mL 0.5 M succinic acid pH 5,20 g Noble agar, and 855 mL deionized water. Following autoclavesterilization, 40 mL sterile 50% glucose and 2 mL 10 mg/mLchloraphenicol were added and plates poured.ura selection media was composed of 6.7 g yeast nitrogen base withammonium sulfate, 5 g casamino acids, 100 mL 0.5 M succinic acid pH 5,and 855 mL deionized water. Following autoclave sterilization, 40 mLsterile 50% glucose and 2 mL 10 mg/mL chloraphenicol were added.YP+10% glucose media was composed of 500 mL YP broth and 100 mL sterile50% glucose.YP broth was composed of 10 g/L of yeast extract, 20 g/L of peptone.YPD plates were composed of 10 g of yeast extract, 20 g of peptone, 20 gbacto agar, and deionized water to 960 mL. Following autoclavesterilization, 40 mL sterile 50% glucose was added and plates poured.TAE was composed of 4.84 g/L of Tris base, 1.14 ml/L of glacial aceticacid, and 2 mL/L of 0.5 M EDTA pH 8.0.TBE was composed of 10.8 g/L of Tris base, 5.5 g/L boric acid, and 4mL/L of 0.5 M EDTA pH 8.0.LiOAc/TE solution was composed of 8 parts sterile water, 1 part 1 MLiOAc, and 1 part 10× TE.10× TE (200 mL) was composed of 2.42 g Tris Base, 4 mL 0.5M EDTA, pH8.0. 5 M HCl was used to adjust the pH to 7.5 and the solution wassterilized by autoclave.PEG/LiOAc/TE Solution was composed of 8 parts 50% PEG3350, 1 part 1 MLiOAc, and 1 part 10× TE.50% PEG3350 was prepared by adding 100 g PEG3350 to 150 mL water andheating and stirring until dissolved. The volume was then brought up to200 mL with water and the sterilized by autoclave.ScD FOA plates were composed of 275 mL 2×-ScD 2×FOA liquid media and 275mL 2×-ScD 2×FOA plate media, melted and cooled to 65° C.2×-ScD 2×FOA liquid media was composed of 6.66 g yeast nitrogen basewithout amino acids, 1.54 g ura-DO supplement (Clontech, Mountain View,Calif., USA), 20 g dextrose, 50 mg uracil, 2 mg uridine, and 2 g 5-FOA(5-fluoroorotic acid, monohydrate; Toronto Research Chemicals, NorthYork, ON, Canada) and water to 1 L. The resulting solution was filteredto sterilize.2×-ScD 2×FOA plate media was composed of 11 g bacto agar and 275 mLwater. The resulting solution was autoclaved to sterilize.DM2 medium was composed of ammonium sulfate (5.0 g/L), magnesium sulfateheptahydrate (0.5 g/L), potassium phosphate monobasic (3.0 g/L), traceelement solution (1 mL/L) and vitamin solution (1 mL/L). Afterdissolving all medium components, the pH of the medium was adjusted tothe desired initial pH using an appropriate base (e.g, KOH).Trace element solution was composed of EDTA (15.0 g/L), zinc sulfateheptahydrate (4.5 g/L), manganese chloride dehydrate (1.0 g/L),Cobalt(II)chloride hexahydrate (0.3 g/L), Copper(II)sulfate pentahydrate(0.3 g/L), disodium molybdenum dehydrate (0.4 g/L), calcium chloridedehydrate (4.5 g/L), iron sulphate heptahydrate (3 g/L), boric acid (1.0g/L), and potassium iodide (0.1 g/L).Vitamin solution was composed of biotin (D−; 0.05 g/L), calciumpantothenate (D+; 1 g/L), nicotinic acid (5 g/L), myo-inositol (25 g/L),pyridoxine hydrochloride (1 g/L), p-aminobenzoic acid (0.2 g/L), andthiamine hydrochloride (1 g/L).DM1 X-α-gal plates were composed of DM1 salts, 20 g/L glucose, traceelement solution, vitamin solution, 2 mL/L X-α-gal (16 mg/mL), 20 g/Lagar.

DM1 salt solution was composed of 2.28 g/L urea, 3 g/L potassiumphosphate monobasic, and 0.5 g/L magnesium sulfate heptahydrate.

Butterfields Phosphate Buffer was composed of 1.25 mL/L of StockSolution (26.22 g/L Potassium Dihydrogen Phosphate and 7.78 g/L SodiumCarbonate) and 5 mL/L of a Magnesium Chloride solution (81.1 g/LMgCl₂-6H₂O). The resulting solution was autoclaved to sterilize, and pHadjusted to 7.2.

CNB1 shake flask media was composed of urea (2.3 g/L), magnesium sulfateheptahydrate (0.5 g/L), potassium phosphate monobasic (3.0 g/L), traceelement solution (1 mL/L) and vitamin solution (1 mL/L), glucose (120.0g/L), 2-(N-Morpholino)ethanesulfonic acid (MES) (97.6 g/L). Afterdissolving all medium components, the pH of the medium was adjusted toan initial pH of 5.8 using an appropriate base (e.g, KOH).

TABLE 0 Primers sequences SEQ Identifier ID NO: Sequence (5′-3′) 0611166152 TAAAACGACGGCCAGTGAATTCCGCGGCGGCCGCGAGTCCATCGGTTCCTGTCA 0611167 153CATAAGAAAATCAAAGACAGAAGGCGCGCCTTTGCTAGCATTTTTGTGTTTTGCT GTGT 0611168 154ACACAGCAAAACACAAAAATGCTAGCAAAGGCGCGCCTTCTGTCTTTGATTTTCTTATG 0611169 155GGGGGAAAGAACTACCAATAGGCCTCCTTTAATTCGGAGAAAATC 0611170 156GATTTTCTCCGAATTAAAGGAGGCCTATTGGTAGTTCTTTCCCCC 0611171 157AAAATAAACTAGTAAAATAAATTAATTAATTATCTAGAGAGGGGGTTATAT 0611172 158ATATAACCCCCTCTCTAGATAATTAATTAATTTATTTTACTAGTTTATTTT 0611173 159GACCATGATTACGCCAAGCTCCGCGGCGGCCGCCCAGTCAAAACCTTCTTCTC 0611174 160TAAAACGACGGCCAGTGAATTCCGCGGCGGCCGCCTTTGAAGGAGCTTGCCA 0611175 161CTATTCCTTCCTCAAATTGCTGTTTAAACGCGTTGAAGATCTATTCTCC 0611176 162GGAGAATAGATCTTCAACGCGTTTAAACAGCAATTTGAGGAAGGAATAG 0611177 163AATGTTCATTTTACATTCAGATGTTAATTAAGGTCTAGATGTGTTTGTTTGTGTG 0611178 164CACACAAACAAACACATCTAGACCTTAATTAACATCTGAATGTAAAATGAACATT 0611179 165GACCATGATTACGCCAAGCTCCGCGGCGGCCGCAATGCCAAGAGTTATGGGGC 0611184 166GGCTACCCTATATATGGTGAGCG 0611185 167 GGGTCCAAGTTATCCAAGCAG 0611186 168CCTTAATTAACCGTAAAGTTGTCTCAATG 0611189 169CAGCAAAACACAAAAATTCTAGAAAATTTAATTAACATCTGAATGTAAAATGAAC 0611191 346CCTTAATTAATTATCCACGGAAGATATGATG 0611195 170GTTCATTTTACATTCAGATGTTAATTAAATTTTCTAGAATTTTTGTGTTTTGCTG 0611196 171GCTCTAGATAAAATGCCCTCTTATTCTGTCGC 0611199 347GCTCTAGATAAAATGTCCCAAGGTAGAAAAGC 0611225 172 GACTGGATCATTATGACTCC0611245 272 CGAACCAATTCAAGAAAACCAAC 0611250 173ACGCCTTGCCAAATGCGGCCGCGAGTCCATCGGTTCCTGTCAGA 0611251 174TCAAAGACAGAATTAATTAAGCTCTAGAATTTTTGTGTTTTGCTGTGTT 0611252 175AACACAAAAATTCTAGAGCTTAATTAATTCTGTCTTTGATTTTCTTATG 0611253 176GTTTAAACCTTTAATTCGGAGAAAATCTGATC 0611254 177GTTAACGGTACCGAGCTCTAAGTAGTGGTG 0611255 178AACCGATGGACTCGCGGCCGCATTTGGCAAGGCGTATCTAT 0611256 179GGCGCCAGCAATTTGAGGAAGGAATAGGAG 0611257 180AAACTATTAGATTAATTAAGCTCGCGATGTGTTTGTTTGTGTGTTTTGTGTG 0611258 181CAAACAAACACATCGCGAGCTTAATTAATCTAATAGTTTAATCACAGCTTAT 0611259 182TACATTATGGTAGCGGCCGCGTGTGACATTTTGATCCACTCG 0611260 183TGGATCAAAATGTCACACGCGGCCGCTACCATAATGTATGCGTTGAG 0611261 184GGGCCCTAAAAGTGTTGGTGTATTAGA 0611263 185GCTAGCTCAACAAACTCTTTATCAGATTTAGCA 0611264 186GCTAGCGAGGAAAAGAAGTCTAACCTTTGT 0611266 187TCGCGATAAAATGTCAACTGTGGAAGATCACTCCT 0611267 188TTAATTAAGCTGCTGGCGCTTCATCTT 0611268 189 TCGCGATAAAATGTCCAGAGGCTTCTTTACTG0611269 190 TTAATTAACTAAAGGTCTCTCACGACAGAG 0611283 191GTTAACCCGGTTTAAACATAGCCTCATGAAATCAGCCATT 0611284 192GGGCCCATATGGCGCCCGGGGCGTTGAAGATCTATTCTCCAGCA 0611295 193GTTTAAACGATTGGTAGTTCTTTCCCCCTC 0611296 194AATAAATTAAGGGCCCTTTATCGCGAGAGGGGGTTATATGTGTAAA 0611297 195TATAACCCCCTCTCGCGATAAAGGGCCCTTAATTTATTTTACTAGTTTAT 0611298 196GTTTAAACTTTTGTAGCACCTCCTGGT 0611376 197CAGCAAAACACAAAAAGCTAGCTAAAATGTTACGTACCATGTTCAAAA 0611377 198GAAAATCAAAGACAGAAGGCGCGCCTTATGCTGTAACAGCCTGCGG 0611378 199CAGCAAAACACAAAAAGCTAGC TAAA ATGTTAAGAACCATGTTCAAATC 0611379 200GAAAATCAAAGACAGAAGGCGCGCC TTATGCAGTAACAGCTTGTGGG 0611552 273TCTGTCCCTTGGCGACGC 0611553 274 CTTTTCAAACAGATAAGTACC 0611554 201GCATGGTGGTGCAAGCGACG 0611555 202 GGTGCTGCATTTGCTGCTG 0611622 275ATGGGCTGACCTGAAAATTC 0611631 203 TGTATACAGGATCGAAGAATAGAAG 0611632 204GAACGTCTACAACGAGGTGAAC 0611661 205 GGCGCGCCTCGCGATAAAATG 0611662 206AGGGCCCTTAATTAATTATGCAGTAACAGCTT 0611717 207 CGCTACGATACGCTACGATA0611718 208 CTCCCTTCCCTGATAGAAGG 0611814 209 GGGGAGCAATTTGCCACCAGG0611815 210 CTCCTTCATTTAACTATACCAGACG 0611816 211GACAGATGTAAGGACCAATAGTGTCC 0611817 212 CCATATCGAAATCTAGCCCGTCC 0611828213 CATAAGAAAATCAAAGACAGAAGGCGCGCCTTTGCTAGCTTTTTGTGTTTTGCTGTGT 0611954348 GCTAGCTAAAATGTTTGGTAATATTTCCCA 0611957 349TTAATTAACTATTTATCTAATGATCCTC 0611997 350TCTAGATAAAATGTCTATTAGTGAAAAATATTTTCCTCAAG 0611998 351TTAATTAACTTTTAAATTTTGGAAAAAGCTTGATCAATAATGG 0612055 214ATTGGACACAACATTATAT 0612150 238 ACGCGTCGACTCGACATTTGCTGCAACGGC 0612151239 CTAGTCTAGATGTTGTTGTTGTTGTCGTT 0612271 215GTAAAACGACGGCCAGTGAATTGTTAACATAGGCTCCAACATCTCG 0612272 216GGAACCGATGGACTCGCGGCCGC GTGGGATATTGGAAG 0612273 217GGAGGTGCTACAAAAGGAATTC 0612274 218GACCATGATTACGCCAAGCTCCGCGGAGTCAAAACCTTCTTCTCTACC 0612275 219GTAAAACGACGGCCAGTGAATTC TTTGAAGGAGCTTGCCAAGAAAC 0612276 220CCTATTCCTTCCTCAAATTGCTG 0612277 221ATAACTCTTGGCATTGCGGCCGCCAAGTTAGTTAGAGC 0612278 222ATGACCATGATTACGCCAAGCTCCGCGGCAAAGACGGTGTATTAGTGCTTG 0612356 223CACAAAACACACAAACAAACACAGCTAGCAAAGGCGCGCCATCTAATAGTTTAATCACAGCTTA 0612357224 GCCGTTGCAGCAAATGTCGAGGCCTGTGTGACATTTTGATCCACTCG 0612358 225CGAGTGGATCAAAATGTCACACAGGCCTCGACATTTGCTGCAACGGC 0612359 226CATTTTACATTCAGATGTTAATTAATTATCTAGATGTTGTTGTTGTTGTCG 0612360 227CGACAACAACAACAACATCTAGATAATTAATTAACATCTGAATGTAAAATG 0612361 228GCTCTAACTAACTTGGCGGCCGCTTTTATTATAAAATTATATATTATTCTT 0612366 276GCTGAAAATATCATTCAGAGCAT 0612367 277 ACTGTTGATGTCGATGCC 0612378 229AACACACAAACAAACACAGCTAGCTAAAATGTTAAGAACTATGTTTA 0612379 230GATTAAACTATTAGATGGCGCGCCTTATGCAGTAACTGCTTGTGGA 0612470 231CGACGGCCAGTGAATTCGTTAACCCGTTTCGATGGGATTCCC 0612471 232CAGGAACCGATGGACTCGCGGCCGCTCCCTTCTCTAAATGGACTGC 0612472 233TATATAATTTTATAATAAAAGCGGCCGCACCAGGGGTTTAGTGAAGTC 0612473 234CATGATTACGCCAAGCTCCGCGGCCATAACTGACATTTATGGTAAGG 0612579 278TCTGAATGCAGTACGAGTTG 0612695 240CAAAACACAGCAAAACACAAAAAGCTAGCATGTATAGAACCTTGATGAG 0612698 242CAAAACACACAAACAAACACAGCTAGCATGTACAGAACGTTAATGTCTGC 0612724 241CAAAGACAGAAGGCGCGCCTTATAAGATGGTTCTCGCTGG 0612725 243GTGATTAAACTATTAGATGGCGCGCCTTACAGGATGGTTCTGGCAGG 0612794 279GAAGGGGGTCCAAGTTATCC 0612795 235 CCAACAATCTTAATTGGTGAC 0612891 236GGCTGTTACCGCCTAATTAA 0612893 237 GTTCTTAACATTTTAGCTAGCTG 0612908 280GATATGGGCGGTAGAGAAGA 0612909 281 GCTCCTTCAAAGGCAACACA 0612910 282TGAACTATCACATGAACGTA 0612911 283 TCAAGGTAGGGTCACTTAAC 0613034 284TGATTCCTTCAATCACAGGT 0613035 285 AACCGACCTATCGAATGCCT 0613178 286ACCATGATTACGCCAAGCTTGGTACCTTGGGGTTTACGCTTACAGCGTACT 0613179 287TCAACGCCCCGGGGGATCTGGATCCGCGGCCGCAAGAAATTCCTTTCTTTTCCCCTTTATA 0613180288 AACCGATGGACTCCTCGAGGGATCCGCGGCCGCGCATAACAAAATTGTGCCTAACCCA 0613181289 ACGACTCACTATAGGGCGAATTGGGCCCCGGGAAAAGGAGAGAGAAAAGGAGA 0613183 290TATGACCATGATTACGCCAAGCTTGGTACCTCCTACAGAAGTAGCACCAGCACCA 0613184 291GGAACCGATGGACTCCTCGAGGGATCCGCGGCCGCAGACTACCGTGTAAATAAGAGTACC 0613185 292TCTTCAACGCCCCGGGGGATCTGGATCCGCGGCCGCATTTGATATAAACGCTTCTATAATA 0613186293 GTAATACGACTCACTATAGGGCGAATTGGGCCCAACATCTGCTGCTGTAATATATTCA 0613241294 GCATGTCTGTTAACTCTCAAACCAT 0613242 295 TCCATAATCCAATAGATCATCCC0613243 296 AACACAATGGAACCAACCTAGT 0613416 297ATTTACACGGTAGTCTGCGGCCGCCATAGCCTCATGAAATCAGCC 0613417 298CTCTAGGTTCACTGGTTGTTTCTTGGGCTGCCTCCTTCAA 0613418 299TTGAAGGAGGCAGCCCAAGAAACAACCAGTGAACCTAGAG 0613419 300TATTATAGAAGCGTTTATATCAAATGCGGCCGCGGATCCAGATCCCCCGGGGCGTT 0613550 301AATGATCAACTTGAGAGGTA 0613551 302 CAGGTCTGTTACATAAAGCA 0613688 303GTTTACGCTCAAATCTCCTCC 0613689 304 GGTACATGAAGCAGGCTTTGAAGG 0613695 305GATTGTGTCCGTCAGCCTTTGCTC 0613746 306 TACCATATTTTCAGAGGATCA 0613747 307AGGATGTTCTTGCCTGCAAGT 0614233 308 GATGATATAGTTGATGCTTTCCAAAG 0614626 344AGGGTACCTTAGTACGAAGG 0614627 345 CTATTCTTACGATGAAGGCG CM647 262AATGATCCATGGTCCGAGAG oACN48 309 GGGCCCCTTCATTTACGAAATAAAGTGCCGCGG oACN49310 GCGGCCGCAAATAAATTTAAAATAAACGATATCAAAATTC oACN50 311CGACGCCAAAGAAGGGCTCAGCCGAAAAG oACN51 312CCATTTCTTTTTCGGCTGAGCCCTTCTTTGGC oACN52 313GCGGCCGCAATAACCTCAGGGAGAACTTTGGC oACN53 314GAGCTCCCAAACAAGGTCTCAGTAGGATCG oACN54 315 CGCCATAAGGAGAGGCTGTAGATTTGTCoACN55 316 CCAGGACATCTTGCTTGCAATGTCG oANN1 317GTTCCATCGGGCCCCTAAAGGTCTCTCACGACAG oANN2 318CAAACACA TCGCGA TAAAATGTCCAGAGGCTTC oANN5 319 GCAAGACCTTGGATCTGAAGGGoANN6 320 CGAACCAATTCAAGAAAACCAACAG oANN7 321GGGCCC GTCCCTTGGCGACGCCCTGATC oANN8 322GCGGCCGCTATTTTTGTGTTTTGCTGTGTTTTG oANN9 323GCGGCCGCC ATCTGAATGTAAAATGAACATTAAAATG oANN10 324GAGCTCCCCCAGTTGTTGTTGCAATTAC oANN11 325 GAAGAGACGTACAAGATCCGCC oANN12326 TAGGAATGGTGCATCATCCAAC oANN13 327TTCTTATCTGAAAACTCCGAGTTCGCAAAGAAGGTTGAAG oANN14 328TTATTGAAATTAATCCAAGGATTCAAGTTGAACATACAATTACTG oANN15 329TTTCCAAAAAAGTTCGTGAGTTCGATGGTTGTATGATTATGG oANN16 330CCTCTACCACCACCACCAAATG oANN20 331 GGGAAGAAACTAAGAAGAAGTATG oCA405 260GCAACTGATGTTCACGAATGCG oCA406 261 TTGCCGTTGCAGCAAATCTC oHJ1 332CAATCCTTCTAGAGAGGGGGTTATATGTGTAAATATAGAGTTTG oHJ2 333TTCTACCCTCGAGATTGGTTCTTTCCCCCTCTCAAG oHJJ116 334GTGGTCAATCTAGAAAATATGACTGACAAAATCTCCCTAGGTAC oHJJ117 335CAATTTTGGAGCTGATTCCAAATCGTAAAC oJLJ28 264 GCACCAAGAGCAGTTTTCCCATCTATTGoJLJ29 265 CCATATAGTTCTTTTCTAACATCAACATCACACTTC oJLJ30 266GGAGAATAGATCTTCAACGCTTTAATAAAGTAGTTTG oJLJ31 267CGTGTTGCGCAATAAAACCAATGAC oJLJ43 336 CAAGAGTATCCCATCTGACAGGAACCGATGGoJLJ44 337 GCTGGAGAATAGATCTTCAACGCCCCG oJLJ45 338CGGAGAAGGCGTATAAAAAGGACACGGAG oJLJ46 339GGATAAAAGTATTACATACGTACAGGATTGTGTATTAGTGTATTTCG oJLJ57 340CCTCCAGTGTTTTTCTCTCTGTCTCTTTGTTTTTTTTTTC oJY11 268 CCTCGAAGAGCTTGAATTTGoJY12 269 GTAGTGAATGTCCGGATAAG oJY13 270 GCAAGGTCATGAGGTTAAAG oJY14 271AACACTTATGGCGTCTCCTC oJY44 341GGTTAATTAATTTATTTGTACATAAAAACCACATAAATGTAAAAGC oJY45 342GAATTCCTTTAATTCGGAGAAAATCTGATCAAGAG WG26 363 ACGGCAGTATACCCTATCAGG

Miscellaneous Sequences

Promoters: The PDC, TDH3, ENO1, and PGK1 promoters described in theExamples were derived from the I. orientalis sequences of SEQ ID NOs:244, 245, 246, and 247, respectively.Terminators: The TAL, TKL, RKI, and PDC terminators described in theExamples were derived from the I. orientalis sequences of SEQ ID NOs:248, 249, 250, and 251, respectively. The URA3 promoter, ORF, andterminator described in the Examples were derived from the I. orientalissequence of SEQ ID NO: 252.

Example 1A: Selection of Host Yeast Cells Based on 3-HP Tolerance

A set of wild-type yeast strains were tested for their ability to growin the presence of 3-HP.

The range of 3-HP concentrations to utilize in primary screeningprocedures was determined by evaluating the ability of seven wild-typeyeast strains (Table 1, set A) to grow on media containing varyinglevels of 3-HP. 3-HP used in these experiments was chemicallysynthesized from an aqueous acrylic acid solution (30%) and a CO₂catalyst at 200 psi and 175° C. as described in WO04/076398. Residualacrylic acid was removed using a countercurrent extraction withisopropyl ether at room temperature (see WO05/021470).

Cells were streaked onto YPD plates and grown overnight. A cell slurrywith an OD₆₀₀ of around 4 was made in YPD media, pH 3.0, and this slurrywas used to inoculate microtiter wells containing various concentrationsof 3-HP (pH 3.9) to an OD₆₀₀ of 0.05. Plates were covered with a gaspermeable membrane and incubated in a 30° C./300 RPM shaker overnight.Optical densities for each well were measured at a wavelength of 600 nmin a GENios model plate reader (Tecan), and plates were observedvisually for growth. The highest 3-HP concentration that one or more ofthe strains grew in (125 g/L) was chosen as the upper range for primaryscreening procedure.

Primary Screening

For the primary screening procedure, 89 wild-type yeast strains werescreened for growth on microtiter plates at 0 g/L, 75 g/L, 100 g/L, or125 g/L 3-HP (pH 3.9) using the same protocol used for range finding. Afresh YPD plate was used for each strain, and a slurry with an OD₆₀₀ ofaround 4 was made in YPD media, pH 3.0. The slurry was used to inoculateeach well to an OD₆₀₀ of 0.05. Plates were covered with a gas permeablemembrane, and incubated in a 30° C./300 RPM shaker overnight. Opticaldensities for each well were measured at a wavelength of 600 nm in aGENios model plate reader, and plates were observed visually for growth.A similar protocol was run to evaluate growth at lactic acidconcentrations of 0 g/L, 30 g/L, 45 g/L, and 60 g/L. Table 1 summarizesthe highest concentration of 3-HP or lactic acid at which growth wasobserved.

Fifteen strains were identified that were capable of growing at 100 g/L3-HP or grew well at 75 g/L 3-HP (Table 1, set B). To further narrow thestrains, a second microtiter plate test was conducted. This test wassimilar to the first, but utilized 3-HP concentrations of 100 g/L, 112.5g/L, and 125 g/L (pH 3.9). From this test, eleven strains wereidentified (Table 1, set C) that grew well at 75 g/L 3-HP or showed somegrowth at both 75 and 112.5 g/L 3-HP. These eleven strains were advancedto the secondary screening. Four strains that had poor growth at 75 g/L3-HP and no growth at 112.5 g/L 3-HP were not advanced to the secondaryscreening. It is expected that strains not advancing to the secondaryscreen would exhibit economically inferior performance in a commercialfermentation process. However, it is possible that one or more suchstrains could nonetheless meet the minimum requirements for acommercially viable fermentation process.

TABLE 1 Growth in 3-HP or lactic acid Primary Primary screen: screen:lactic Culture 3-HP acid Yeast strain collection # Set (g/L) (g/L)Bulleromyces albus ATCC 96272 No NG growth at 0 g/L (NG) Candida blankiiATCC 18735 0 30 Candida boidinii PYCC 70-104 0 30 Candida catenulataATCC 20117 0 NG Candida etchellsii PYCC 60-8 0 30 Candida famata ATCC20284 0 0 Candida fluviatilis ATCC 38623 0 0 Candida geochares Cargill1978 75 60 Candida guilliermondii ATCC 20118 0 0 Candida intermedia ATCC20178 0 0 Candida kefyr ATCC 44691 75 30 Candida lactiscondensi ATCC96927 0 NG Candida lambica ATCC 38617 B, C 75 45 Candida ATCC 20361 0 NGmethanosorbosa Candida milleri ATCC 60591 0 30 Candida milleri ATCC60592 0 45 Candida naeodendra ATCC 56465 0 NG Candida parapsilosis ATCC20179 0 0 Candida pignaliae ATCC 36592 0 NG Candida pseudolambica ATCC96309 0 0 Candida rugosa ATCC 20306 0 45 Candida shehatae NCYC 2389 0 0Candida sonorensis ATCC 32109 A 0 30 Candida sorbophila Cargill 1973 7545 Candida sorboxylosa ATCC 24120 0 30 Candida sorosivorans ATCC 38619 B100 60 Candida tenuis MUCL 47216 or 0 NG MUCL 31253B Candida valida ATCC28525 B 75 45 Candida vanderwaltii MUCL 300000 0 45 Candida zemplininaPYCC 04-501 A, B, C 100 60 Candida zeylanoides ATCC 20347 75 45Citeromyces matritensis ATCC 34087 0 NG Debaryomyces castellii PYCC70-1022 0 30 Debaryomyces hansenii ATCC 90624 0 NG Debaryomyces ATCC20280 0 0 polymorphus Dekkera anomala ATCC 20277 0 0 Dekkera lambicaATCC 10563 0 0 Hyphopichia burtonii ATCC 20030 0 0 Issatchenkiaorientalis ATCC 24210 B, C 100 45 Issatchenkia orientalis ATCC 60585 B,C 100 45 Issatchenkia orientalis ATCC PTA-6658 A, B, C 100 60Issatchenkia orientalis CD1822 (Cargill A, B, C 100 60 collection - seedescription below) Kluyveromyces lactis ATCC 8585 A 0 0 KluyveromycesATCC 52486 A 75 45 marxianus Kluyveromyces ATCC 52709 0 30thermotolerans Kodamaea ohmeri ATCC 20282 0 45 Kluyveromyces yarrowiiATCC 36591 NG NG Lipomyces starkeyi ATCC 12659 0 0 Lipomyces tetrasporusATCC 56306 NG NG Metschnikowia ATCC 9889 0 0 pulcherrima Myxozymakluyveri ATCC 76214 NG NG Nematospora coryli ATCC 20292 0 NG Pachysolentannophilus NCYC 614 0 30 Pichia anomala ATCC 2102 0 0 Pichia fermentansATCC 28526 0 30 Pichia fluxuum ATCC 28778 NG NG Pichia jadinii ATCC 99500 0 Pichia membranifaciens NCYC 2696 B, C 125 60 Pichia nakasei ATCC24116 0 0 Pichia silvicola ATCC 16768 0 0 Pichia stipitis CBS 6054 0 0Pichia strasburgensis ATCC 34024 0 0 Pichia tannicola ATCC 2261 0 0Pichia toletana ATCC 58362 NG NG Saccharomyces ATCC 96784 75 30cerevisiae Saccharomyces ATCC 90739 0 45 bayanus Saccharomyces bulderiMYA-402 B, C 100 60 Saccharomyces bulderi MYA-404 B, C 100 45Saccharomyces ATCC 20033 0 0 capsularis Saccharomyces CEN-PK 113-7D A, B100 45 cerevisiae Saccharomyces ludwigii NCYC 734 75 45 SaccharomycopsisMUCL 44417 0 0 crataegensis Saccharomycopsis MUCL 31237 0 45 javensisSaccharomycopsis vini NRRL Y-7290 0 0 Saccharomyces uvarum ATCC 76514 00 Schizosaccharomyces ATCC 10660 0 0 japonicus Schizosaccharomyces NCYC535 B, C 100 60 pombe Torulaspora delbrueckii ATCC 52714 75 0Torulaspora pretoriensis ATCC 42479 B 75 0 Wickerhamiella CBS 8452 0 30occidentalis Yamadazyma ATCC 90197 0 0 guilliermondii Yamadazymahaplophila ATCC 20321 0 0 Yamadazyma stipitis ATCC 201225 0 0 Yarrowialipolytica ATCC 46330 0 45 Zygosaccharomyces ATCC 36946 75 45 bailiiZygosaccharomyces NCYC 3134 0 45 bisporus Zygosaccharomyces NCYC2897 060 kombuchaensis Zygosaccharomyces NCYC 2928 B, C 100 45 lentusZygosaccharomyces ATCC 34890 75 30 rouxii

I. orientalis strain CD1822 tested above was generated by evolving I.orientalis ATCC PTA-6658 for 91 days in a glucose limited chemostat. Thesystem was fed with 15 g/L dextrose in a DM medium, and operated at adilution rate of 0.06 h⁻¹ at pH=3 with added lactic acid in the feedmedium. The conditions were maintained with a low oxygen transfer rateof approximately 2 mmol L⁻¹h⁻¹, and dissolved oxygen concentrationremained constant at 0% of air saturation. Single colony isolates fromthe final time point were characterized in two shake flask assays. Inthe first assay, the strains were characterized for their ability toferment glucose to ethanol in the presence of 25 g/L total lactic acidwith no pH adjustment in the DM1 defined medium. In the second assay,the growth rate of the isolates were measured in the presence of 25, 32and 45 g/L of total lactic acid, with no pH adjustment in DM1 definedmedium. Strain CD1822 was a single isolate selected based on themeasured fermentation rates and growth rates. Other methods for evolvingI. orientalis include, but are not limited to, repeated serialsubculturing under the selective conditions as described in e.g., U.S.Pat. No. 7,629,162. Such methods can be used in place of, or in additionto, using the glucose limited chemostat method described above. As canbe appreciated by one of skill in the art, strains could be generatedusing a similar evolution procedure in the presence of added 3-HP ratherthan lactic acid to develop improved 3-HP tolerance. Additionally,strains could be mutagenized prior to selection, as described herein(e.g., see Example 1B).

Secondary Screening

For the first part of the secondary screen, growth rates were measuredat pH 3.9 in YPD media containing 0 g/L, 35 g/L, or 75 g/L 3-HP. Shakeflasks were inoculated with biomass harvested from seed flasks grownovernight to an OD₆₀₀ of 6 to 10. 250 mL baffled growth rate flasks (50mL working volume) were inoculated to an OD₆₀₀ of 0.1 and grown at 250rpm and 30° C. Samples were taken throughout the time course of theassay and analyzed for biomass growth via OD₆₀₀. The resulting OD₆₀₀data was plotted and growth rates were established.

TABLE 2 Growth rate (μ) in 3-HP Strain 0 g/L 3-HP 35 g/L 3-HP 75 g/L3-HP Issatchenkia orientalis 0.56 0.51 0.29 ATCC 60585 Issatchenkiaorientalis 0.62 0.52 0.28 CD1822 Candida lambica 0.65 0.53 0.30 ATCC38617 Candida valida 0.51 0.32 0.14 Issatchenkia orientalis 0.61 0.530.32 PTA-6658 Issatchenkia orientalis 0.58 0.51 0.26 24210 Saccharomycesbulderi 0.53 0.45 0.28 MYA 402 Pichia membranifaciens 0.41 0.39 0.32Saccharomyces bulderi 0.55 0.44 0.27 MYA 404 Schizosaccharomyces pombe0.41 0.35 0.21 Zygosaccharomyces lentus 0.61 0.41 0.20

For the second part of the secondary screen, glucose consumption wasmeasured for the same ten strains at pH 3.9 in YPD media containing 100g/L glucose and 0 g/L, 35 g/L, or 75 g/L 3-HP. Shake flasks wereinoculated with biomass harvested from seed flasks grown overnight to anOD₆₀₀ of 6 to 10. 250 mL baffled glycolytic assay flasks (50 mL workingvolume) were inoculated to an OD₆₀₀ of 0.1 and grown at 250 RPM and 30°C. Samples were taken throughout the time course of the assay andanalyzed for glucose consumption using a 2700 Biochemistry Analyzer fromYellow Springs Instruments (YSI). The resulting data was plotted andglucose consumption rates were established.

TABLE 3 Glucose consumption rate (g/L/hr) in 3-HP Strain 0 g/L 3-HP 35g/L 3-HP 75 g/L 3-HP Issatchenkia orientalis 5.5 4.2 3.3 ATCC 60585Issatchenkia orientalis 5.5 4.2 4.2 CD1822 Candida lambica 4.2 4.2 3.5ATCC 38617 Candida valida 5.5 2.2 2.1 Issatchenkia orientalis 5.5 4.24.1 PTA-6658 Issatchenkia orientalis 24210 4.2 4.2 3.8 Saccharomycesbulderi 4.2 4.2 4.0 MYA 402 Pichia membranifaciens 0.4 2.1 1.2Saccharomyces bulderi 4.2 4.2 3.8 MYA 404 Schizosaccharomyces pombe 2.53.1 2.0 Zygosaccharomyces lentus 3.4 0.8 0.3

Four of the strains (P. membranifaciens, S. pombe, C. valida, and Z.lentus) did not achieve the 2.5 g/L/hr glucose utilization rate underthe 75 g/L 3-HP (pH 3.9) conditions that would be required for aneconomic fermentation process.

To identify the leading strains in 3-HP, strain performance was gradedin three categories. Two of these categories were based on differentaspects of growth rate: 1) growth rate at highest acid concentration and2) slope of the growth rates plotted against acid concentration. Thethird category was the glycolytic rate at the highest acidconcentration. This grading was done on a normalized scale using thehighest and lowest value for each rating as the normalized boundaries.Each strain thus received a grade between 0 and 1 for each category,with 1 being the highest possible score. The overall rating of a strainwas the sum of the normalized value for the three categories. A weightedscore was made in which the growth rate and glycolytic rate were equallyweighted. In this case the glycolytic rate at the highest acidconcentration was weighted at 50%, while the two growth rate ratingswere weighted at 25% each. Normalized values per category and sum andweighted scores are summarized in Table 4.

TABLE 4 Strain grades in 3-HP Growth rate @ 75 g/ Growth L 3- rateGlycolic Sum Weighted Strain HP slope rate Score score Issatchenkiaorientalis 1.00 0.37 0.97 2.34 0.83 ATCC PTA-6658 Saccharomyces 0.780.49 0.94 2.21 0.79 bulderi MYA 402 Issatchenkia orientalis 0.78 0.211.00 1.99 0.75 CD1822 Saccharomyces 0.72 0.42 0.90 2.04 0.74 bulderi MYA404 Issatchenkia orientalis 0.83 0.44 0.77 2.04 0.70 ATCC 60585Issatchenkia orientalis 0.67 0.28 0.90 1.85 0.69 24210 Candida lambica0.89 0.19 0.82 1.90 0.68 ATCC 38617 Pichia 1.00 1.00 0.23 2.23 0.62membranifaciens Schizosaccharomyces 0.39 0.65 0.47 1.51 0.50 pombeCandida valida 0 0.14 0.40 0.54 0.24 Zygosaccharomyces 0.33 0 0 0.330.08 lentus

Of the strains tested, strains from the species I. orientalis, C.lambica, and S. bulderi showed the greatest potential as productionhosts for 3-HP at low pH.

The same procedures were utilized to screen, rate, and score theoriginal 91 wild-type yeast strains from the primary screen with mediacontaining 0, 25, and 50 g/L lactic acid at pH 2.85 (˜80% free acid).Normalized values and weighted and summed scores were derived for 13strains that were advanced to the secondary screen.

TABLE 5 Strain grades in lactic acid Growth rate Growth Weight- @ 50 g/Lrate Glycolic Sum ed Strain lactic acid slope rate Score score Candidalambica 0.92 1.00 1.00 2.92 0.98 ATCC 38617 Issatchenkia orientalis 0.940.95 1.00 2.89 0.97 ATCC PTA-6658 Issatchenkia orientalis 1.00 0.86 1.002.86 0.97 CD1822 Issatchenkia orientalis 0.89 0.73 1.00 2.62 0.91 24210Candida zemplinina 0.22 0.95 1.00 2.17 0.79 Saccharomyces 0.47 0.45 1.001.92 0.73 bulderi MYA 404 Saccharomyces 0.08 0.91 0.96 1.95 0.73 bayanusSaccharomyces 0.50 0.23 1.00 1.73 0.68 bulderi MYA 402 Candida milleri 00.64 0.92 1.56 0.62 Candida sorosivorans 0.28 0.95 0.59 1.82 0.60Kodamaea ohmeri 0.42 0 0.76 1.18 0.49 Candida geochares 0.17 0.27 0.691.13 0.46 Saccharomyces 0.11 0.68 0 0.79 0.20 javensis

For lactic acid only S. javensis did not achieve the 2.5 g/L/hr glucoseutilization rate at pH 2.85 in media with 50 g/L lactic acid. While I.orientalis, C. lambica, and S. bulderi showed acid tolerance for both3-HP and lactic acids, there were a number of strains that were tolerantfor only one of the acids. This can also be seen in the results of theprimary screen (Table 1). For example, C. milleri, C. rugosa, C.vanderwaltii, K. ohmeri, S. bayanus, S. javensis, Y. lipolytica, Z.bisporus, and Z. kombuchaensis all demonstrated growth at 45-60 g/Llactic acid but no growth at even the lowest concentration of 3-HPtested (35 g/L). Thus, the tolerance of a strain to one organic acidcannot definitively be used as a predictor of its tolerance for otheracids. This is further highlighted by comparing the strains that showed3-HP resistance above with the list of eight strains identified aspreferred hosts for organic acid production in WO03/049525. While two ofthose strains (C. diddensiae and C. entomophila) could not be obtainedfor testing, the other six were included in the primary screen describedabove. Of these six, only C. krusei (tested as I. orientalis) was ableto grow in the presence of 35 g/L 3-HP.

Example 1B: Mutagenesis and Selection of Mutant Strains HavingResistance to 3-HP

Yeast cells selected in Example 1A are subjected to mutagenesis andexposed to selection pressure in order to identify mutants with high3-HP tolerance.

For example, yeast cells from a fresh YP (yeast extract/peptone)+20 g/Lglucose plate or liquid culture (OD₆₀₀ 1-4) are resuspended in sterilewater to an OD₆₀₀ of around 10. 200 μL aliquots of this cell suspensionare pipetted into individual tubes and exposed to 3 μL ethane methylsulfonate (EMS) for approximately one hour, which kills around 65% ofthe cells. Higher EMS concentrations can also be used to increase thekill rate. After exposure, cells are neutralized with 5% sodiumthiosulfate, washed in PBS buffer, recovered in rich media forapproximately four hours, and cultured on selective media. Mock samples(no EMS) are also run to ensure that the conditions are selective.Alternatively, cells can be mutagenized using UV irradiation.

To select for 3-HP resistant mutant strains, aliquots of the EMS-treatedcell suspension (approximately 2×10⁸ of mutagenized cells) are platedonto a potato dextrose agar (PDA) or another media containing 3-HP at alevel at which the parental strain does not grow or grows very slowly.These plates are incubated for several days until colonies appear.Single colonies are purified, streaked on non-selective media toeliminate any adaptive effects of the selection, and re-tested onselective media to confirm increased resistance. Resistant strains arethen tested in a shake flask format with periodic sampling for HPLCanalysis of products and substrates. Alternatively, selection for 3-HPtolerance may be done by chemostat or serial shake flask evolution.Additional rounds of mutagenesis and selection can be performed.Mutagenesis can be used to increase the resistance of a host that doesnot natively meet 3-HP production requirements so that it has thenecessary attributes for commercial 3-HP production.

Example 2A: Procedure for Transformation of DNA into the Host Genome

DNA transformation into the yeast host genome to generate the modifiedyeast strains described in the following examples was conducted based onthe specific procedure below.

Four mL of YP+10% glucose media was added to a 14 mL Falcon tube and thedesired strain was inoculated into this media using a sterile loop. Theculture was grown with shaking at 250 rpm overnight (˜16 hr) at 37° C. 1mL of the overnight culture was added to a 250 mL baffled flaskcontaining 50 mL of liquid YP+10% glucose media. The flask was grownwith shaking at 250 rpm at 37° C. SmaIl aliquots of the culture werewithdrawn at approximately hourly intervals and the OD₆₀₀ was measured.The culture was grown until the OD₆₀₀ was 0.6-1.0.

The cells were harvested by centrifugation at 2279×g at roomtemperature, the pellet was resuspended in 25 mL sterile water, thencentrifuged at 2279×g at room temperature. The pellet was resuspended in1 mL sterile water, and the resuspended cells were transferred to a 1.5mL tube and then pelleted at 16,100×g. The cells were resuspended in 1mL LiOAc/TE solution and then pelleted at 16,100×g. The cell pellet wasthen resuspended in 500 μL LiOAc/TE solution.

The following components were added to a 1.5 mL tube: 100 μL of theabove cells, 10 μL freshly boiled then iced salmon sperm DNA (AgilentTechnologies, Santa Clara, Calif., USA), and 10 μL of the desired,linearized transforming DNA. A control reaction with water instead ofDNA was also prepared. To each transformation reaction, 600 μL ofPEG/LiOAc/TE Solution was added and the reaction incubated on its sideat 30° C. on a 250 rpm shaker platform for 30 minutes. 40 μL DMSO andwas added to each reaction and then incubated in a 42° C. water bath for5 minutes. Cells were pelleted at 5,400×g for 1 min. Cells wereresuspended in water, split in two, and each half of the transformationreaction was plated to a ura selection media plate. Plates were placedat 37° C. Colonies were generally visible after 18 to 24 hr growth,depending on strain background.

A sterile loop was used to transfer a small amount of yeast from a petridish to a 1.5 mL tube containing 300 μL Yeast Lysis Solution (EPICENTRE®Biotechnologies, Madison, Wis., USA) and the genomic DNA was extractedusing the MasterPure™ Yeast DNA Purification Kit (EPICENTRE®Biotechnologies) according to the manufacturer's instructions.

Genomic DNA prepared using the MasterPure™ Yeast DNA Purification Kit(EPICENTRE® Biotechnologies) was used in PCR reactions to determine ifthe correct integration event had occurred in the isolated transformant.A PCR reaction (25 μL) contained 0.5 μL genomic DNA for the strain to bescreened, 1× Crimson Taq™ Reaction Buffer (New England Biolabs, Ipswich,Mass., USA), 25 pmol each of the sense and anti-sense primers, 200 μMeach of dATP, dCTP, dGTP, and dTTP, and 0.625 units of Crimson Taq™ DNApolymerase (New England Biolabs). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific, Westbury, N.Y., USA) programmed forone cycle at 95° C. for 30 seconds followed by 30 cycles each at 95° C.for 30 seconds, 50° C. for 30 seconds, and 68° C. for 1 minute per kbpof the largest expected product, with a final extension at 68° C. for 10minutes. Following thermocycling, the PCR reaction products wereseparated by 1% agarose gel electrophoresis in TAE or TBE buffer and thesizes of the bands visualized and interpreted as for the specificprimers sets as described.

Example 2B: Selection of Insertion Sites

Suitable insertion sites for incorporating exogenous genes into hostyeast cells may be loci for genes that have beneficial or neutraleffects on 3-HP production when deleted in the yeast host cell.Non-limiting examples of suitable insertion sites for selected yeaststrains are described in the working examples herein. One skilled in theart can easily apply the teachings herein for use of these and otherinsertions sites, for example, loci for one or more PDC (e.g., I.orientalis PDC gene encoding the amino acid sequence set forth in SEQ IDNO: 49 and/or comprising the coding region of the nucleotide sequenceset forth in SEQ ID NO: 48), ADH (e.g., I. orientalis ADH gene encodingthe amino acid sequence set forth in SEQ ID NOs: 106, 108, or 110 and/orcomprising the coding region of the nucleotide sequence set forth in SEQID NOs: 105, 107, or 109), GAL6 (e.g., I. orientalis GAL6 gene encodingthe amino acid sequence set forth in SEQ ID NO: 112 and/or comprisingthe coding region of the nucleotide sequence set forth in SEQ ID NO:111), CYB2A (e.g., I. orientalis CYB2A gene encoding the amino acidsequence set forth in SEQ ID NO: 114 and/or comprising the coding regionof the nucleotide sequence set forth in SEQ ID NO: 113), CYB2B (e.g., I.orientalis CYB2B gene encoding the amino acid sequence set forth in SEQID NO: 116 and/or comprising the coding region of the nucleotidesequence set forth in SEQ ID NO: 115), GPD (e.g., I. orientalis GPD geneencoding the amino acid sequence set forth in SEQ ID NO: 118 and/orcomprising the coding region of the nucleotide sequence set forth in SEQID NO: 117), ALD (e.g., I. orientalis ALD homolog gene 5680 encoding theamino acid sequence set forth in SEQ ID NO: 120 and/or comprising thecoding region of the nucleotide sequence set forth in SEQ ID NO: 119, I.orientalis ALD homolog gene 42026 encoding the amino acid sequence setforth in SEQ ID NO: 122 and/or comprising the coding region of thenucleotide sequence set forth in SEQ ID NO: 121, I. orientalis ALDhomolog gene 42426 encoding the amino acid sequence set forth in SEQ IDNO: 124 and/or comprising the coding region of the nucleotide sequenceset forth in SEQ ID NO: 123, or I. orientalis ALD homolog gene 42727encoding the amino acid sequence set forth in SEQ ID NO: 126 and/orcomprising the coding region of the nucleotide sequence set forth in SEQID NO: 125), or PCK (e.g., I. orientalis PCK gene encoding the aminoacid sequence set forth in SEQ ID NO: 128 and/or comprising the codingregion of the nucleotide sequence set forth in SEQ ID NO: 127) genes orhomologs thereof. Where sequences for these genes are unpublished, theycan be obtained using standard procedure such as genome sequencing,probe hybridization of genomic or cDNA libraries, or amplification ofgene fragments using degenerate primers based on known homolog sequence,followed by genome walking to obtain the full sequence. Other suitablelocations for insertion sites include intergenic regions that do notcontain open reading frames.

Example 2C: Techniques for Insertion Vectors, Selection MarkerCassettes, Gene Expression Cassettes, and Integration Constructs

Insertion site vectors are generated for integrating one or moreexogenous genes into host yeast cells. The host yeast cells may be cellsthat have undergone a selection process as described in Example 1, orthey may be cells that have not undergone mutagenesis and/or selection.

To generate insertion site vectors, a region upstream (5′) and a regiondownstream (3′) of the desired insertion site are both amplified usinghost genomic DNA as template. The upstream region is preferably greaterthan 70 bp and less than 1.5 kbp. The resultant target sequences areligated into a cloning vector either simultaneously or sequentially toobtain a vector with one copy of each fragment so that the fragments arecontiguous or nearly contiguous. A unique restriction site may beincorporated between the fragments to allow for insertion of geneexpression cassettes and/or selection marker cassettes. Uniquerestriction enzyme sites may also be incorporated at or near the 5′ endof the upstream fragment and at or near the 3′ end of the downstreamfragment to allow for later removal of the DNA between these sites fromthe cloning vector.

Selection marker cassettes for incorporation into insertion site vectorsare generated using standard cloning techniques. These selection markercassettes contain a gene for a selectable marker, and may also containan upstream promoter and/or a downstream terminator sequence. Examplesof suitable selection marker genes include the URA3, TRP1, HIS, MEL5,CYB2A, LEU2, and G418 genes. Flanking sequences may be incorporated intothe cassette on either side of the promoter/marker gene/terminatorsequences to allow for future loss of the marker through recombination.These flanking sequences may include a direct or inverted repeatedsequence (either functional or nonfunctional sequence) or one or moreIoxP sites.

Gene expression cassettes are generated using standard cloningtechniques. These gene expression cassettes contain the gene to beover-expressed, and may also contain an upstream promoter and/or adownstream terminator sequence. In certain embodiments, two or morecopies of these promoter/gene/terminator combinations may beincorporated into a single gene-expression cassette. Heterologous genesmay be codon-optimized for improved expression in the host yeast strain.A selection marker cassette as described in herein can be cloned intothe gene expression cassette such that it is contiguous or nearlycontiguous with the gene to be over-expressed and any associatedpromoter and/or terminator.

Alternatively, for replacement of native promoters with an exogenouspromoter, the expression cassette may have the selection cassetteupstream of the promoter to be integrated, in between targetingsequences.

Gene expression cassettes can be inserted between the two target sitesequences in the insertion site vectors described herein using standardcloning techniques to generate gene expression integration constructs.One or more selection marker cassettes may also be inserted between thetarget sequences, either as part of the gene expression cassette orseparately. In certain variations, pieces of the gene expressioncassette can be cloned into different insertion site vectors so thatthere is an over-lapping fragment in common between the integrationfragments. For example, one vector might contain an upstream insertionfragment, a promoter, a gene, and a terminator and the second vectormight contain the terminator, selection marker cassette, and downstreaminsertion fragment. In another example, to allow simultaneous insertionof two genes, one vector could contain the upstream insertion fragment,a promoter, a gene, terminator and all or part of a selection markercassette, and the second vector might contain all or part of theselection marker cassette, a second promoter, gene, terminator, and thedownstream insertion fragment.

To generate gene knockout constructs, the insertion site vectors aremade using target DNA sequences derived from the upstream and downstreamflanking regions of the gene to be deleted or disrupted. The selectedtarget sequences may include upstream and downstream flanking regions ofa target gene and/or all or a portion of the target gene coding sequenceor its regulatory elements (e.g., promoter or terminator). One or moreselection marker cassettes may be incorporated into the insertion sitevector between the two target sequences. Where the knockout is to becoupled with expression of an exogenous gene, one or more geneexpression cassettes are also incorporated into the insertion sitevector.

DNA fragments to be integrated into a host yeast genome can belinearized by restriction enzyme digest of the fragment from a cloningvector, or of overlapping fragments from multiple vectors.Alternatively, linear integration fragments can be generated using PCR,or a combination of PCR and restriction enzyme digest. The insertionsite flanking regions can be incorporated into the integration fragmenteither by their presence in the vector template or by incorporation intothe amplification primers. In the latter case, a minimum of 70nucleotides of a flanking region is preferably incorporated into aprimer.

Non-limiting examples of suitable insertion vectors, selection markercassettes, gene expression cassettes, and integration constructs forselected yeast strains are described in the following working examples.One skilled in the art can easily apply the teachings from theseexamples and the preceding specification to generate alternativemodified yeast strains that produce 3-HP.

Example 2D: Construction of Insertion Vector for Expressing an ExogenousGene at the Adh1202 Locus

The plasmid pMIBa107 was created to allow integration of a single geneat the I. orientalis adh1202 locus under the control of the PDC promoterand terminator using URA3 as a selectable marker. The PDC promoter andterminator with the ura selectable marker were PCR amplified and clonedinto pCR4™4BLUNT TOPO® (Invitrogen, La Jolla, Calif., USA) as describedbelow. The PCR fragment containing the PDC promoter, terminator and URA3selectable marker was constructed by SOE PCR. The PDC promoter wasamplified with a primer that contains homology to the PDC terminator onthe 3′ end of the PCR product and the PDC terminator and URA3 selectablemarker were amplified using a primer with homology to the PDC promoteron the 5′ end of the product. These two fragments were then put togethervia SOE PCR.

The PDC promoter was amplified from pACN5 (FIG. 19) using primers0611184 and 0611195. Primer 0611184 introduces a NotI restriction siteto the 5′ end of the PCR product. Primer 0611195 introduces an XbaIrestriction site after the PDC promoter and introduces homology to thePDC terminator on the 3′ end of the PCR product.

The amplification reactions were performed using Platinum® Pfx DNApolymerase (Invitrogen) according to manufacturer's instructions. EachPCR reaction contained 0.5 μL of diluted pACN5 (FIG. 19), 25 μM each ofprimers 0611184 and 0611195, 1× Pfx amplification buffer (Invitrogen), 2mm MgSO₄, 0.2 mM dNTP mix, 1.25 Units Platinum® Pfx DNA polymerase(Invitrogen) in a final volume of 50 μL. The amplification reactionswere incubated in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for 1 cycle at 95° C. for 2 minutes; 30 cycles each at 95° C.for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes; with afinal extension at 72° C. for 3 minutes.

The PCR product was purified by 1% agarose gel electrophoresis using 89mM Tris base-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer. Afragment of approximately 700 bp was excised from the gel and extractedfrom the agarose using a QIAQUICK® Gel Extraction Kit (Qiagen, Valencia,Calif., USA).

The PDC terminator and URA3 selectable marker were amplified from pHJJ76(FIG. 24) using primers 0611189 and 0611185. Primer 0611189 introduceshomology to the PDC promoter on the 5′ end of the PCR product and a PacIrestriction site directly in front of the PDC terminator. Primer 0611185introduces a Not restriction site to the 3′ end of the PCR product. Theamplification reactions were performed using Platinum® Pfx DNApolymerase (Invitrogen) according to manufacturer's instructions. EachPCR reaction contained 0.5 μL of diluted pHJJ76, 25 pM each of primers0611189 and 0611185, 1× Pfx amplification buffer (Invitrogen), 2 mmMgSO₄, 0.2 mM dNTP mix, 1.25 Units Platinum® Pfx DNA polymerase(Invitrogen) in a final volume of 50 μL. The amplification reactionswere incubated in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for 1 cycle at 95° C. for 2 minutes; 30 cycles each at 95° C.for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes; with afinal extension at 72° C. for 3 minutes.

The PCR product was purified by 1% agarose gel electrophoresis using 89mM Tris base-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer. Afragment of approximately 2000 bp was excised from the gel and extractedfrom the agarose using a QIAQUICK® Gel Extraction Kit (Qiagen).

The 2000 bp PDC terminator and URA3 selectable marker PCR product andthe 700 bp PDC promoter PCR product were fused using SOE-PCR. Theamplification reactions were performed using Platinum® Pfx DNApolymerase (Invitrogen) according to manufacturer's instructions. EachPCR reaction contained 8 ng of 2000 bp PDC terminator and URA3selectable marker PCR product, 24 ng of the 700 bp PDC promoter PCRproduct, 50 pM each of primers 0611184 and 0611185, 1× Pfx amplificationbuffer (Invitrogen), 2 mm MgSO₄, 0.2 mM dNTP mix, 2.5 Units Platinum®Pfx DNA polymerase (Invitrogen) in a final volume of 100 μL. Theamplification reactions were incubated in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for 1 cycle at 95° C. for 2 minutes;30 cycles each at 95° C. for 30 seconds, 55° C. for 30 seconds, and 68°C. for 3 minutes; and 1 cycle at 68° C. for 3 minutes.

The 2700 bp PCR product was purified by 1% agarose gel electrophoresisusing 89 mM Tris base-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer.A fragment of approximately 2700 bp was excised from the gel andextracted from the agarose using a QIAQUICK® Gel Extraction Kit(Qiagen).

The 2700 bp PCR product was cloned into pCR™4BLUNT TOPO® (Invitrogen)vector using the Zero Blunt® TOPO® PCR cloning kit for sequencing(Invitrogen) according to the manufacturer's instructions. In a totalreaction volume of 6 μL either 1 or 4 μL of the 2700 bp PCR product, 1μL salt solution (Invitrogen) and 1 μL pCR™4BLUNT TOPO® (Invitrogen)were incubated together at room temperature for 15 minutes. 2 μL of eachcloning reaction was transformed into One Shot® TOP10 ChemicallyCompetent E. coli (Invitrogen) cells according to manufacturer'sinstructions. Transformants were plated onto 2× YT+amp plates andincubated at 37° C. overnight. Several of the resulting transformantswere screened for proper insertion of the desired PCR product by NotIdigestion. A clone yielding the desired band sizes was confirmed to becorrect by DNA sequencing and designated pMIBa100.

The plasmid pHJJ76 (FIG. 24) contains homology to allow gene integrationat the adh1202 locus. Plasmid pHJJ76 was digested with NotI to removethe URA3 selectable marker present inside of the adh1202 homologysequences. The digested pHJJ76 was purified by 1% agarose gelelectrophoresis using 89 mM Tris base-89 mM Boric Acid-2 mM disodiumEDTA (TBE) buffer. A 5.2 kbp fragment was extracted from the agaroseusing a QIAQUICK® Gel Extraction Kit (Qiagen), and then ligated backtogether using T4 DNA ligase. The ligation products were transformedinto One Shot® TOP10 Chemically Competent E. coli (Invitrogen) cellsaccording to manufacturer's instructions. Transformants were plated onto2× YT+amp plates and incubated at 37° C. overnight. Several resultingtransformants were screened by ApaI and SacI digestion. A clone yieldingthe desired digestion products was designated pHJJ76-no ura.

The PDC promoter and terminator and URA3 selectable marker from pMIBa100(supra) was cloned into pHJJ76-no ura to create a plasmid where a genecould be placed under the control of the PDC promoter and terminator forintegration at adh1202. pHJJ76-no ura was digested with NotI followed bytreatment with CIP. The linear 5.2 kbp fragment was purified using aQIAQUICK® PCR Purification Kit (Qiagen). pMIBa100 was digested with NotIand run on a 1% agarose gel using 89 mM Tris base-89 mM Boric Acid-2 mMdisodium EDTA (TBE) buffer. A 2742 bp fragment was excised from the gel,extracted using a QIAQUICK® Gel Extraction Kit (Qiagen), and thenligated into the 5.2 kbp fragment of pHJJ76-no ura using T4 DNA Ligase.The ligation products were transformed into One Shot® TOP10 ChemicallyCompetent E. coli (Invitrogen) cells according to manufacturer'sinstructions. Transformants were plated onto 2× YT+amp plates andincubated at 37° C. overnight. Several of the resulting transformantswere screened by KpnI and XbaI digestion. A clone yielding the desireddigestion products was designated pMIBa107 (FIG. 2).

Example 2E: Construction of Insertion Vector Fragments for ExpressingMultiple Exogenous Genes at the PDC Locus

The following insertion vector fragments can be used to generate adesigned DNA construct that replaces an endogenous I. orientalis PDCgene with a cassette that expresses multiple genes, e.g., three genesdescribed herein expressed from the PDC, ENO1, and TDH3 promoters.Homologous recombination between the left construct (pMhCt068 andderivatives) and the right construct (pMhCt069 and derivatives) resultsin expression of the URA3 protein, resulting in conversion of the strainfrom uracil auxotrophy to uracil prototrophy, allowing for selection ofdesired integrants. The 5′ end of each left-hand construct is homologousto the DNA upstream of the PDC locus, while the 3′ end of eachright-hand construct is homologous to the DNA downstream of the PDClocus. These homologous regions serve to target the expression cassetteto the PDC locus. This targeting approach is depicted schematically inFIG. 3, and can be modified to use any combination of multiple genesdescribed herein to target any suitable locus, e.g., any locus describedabove, such as an ADH locus (see example below) or an ALD locus.

Construction of a Left-Hand Fragment

An empty vector left-hand construct, pMhCt068, was cloned in multiplesteps as described below.

A PCR fragment containing the PDC promoter region and desired additionalrestriction sites and flanking DNA was amplified from genomic I.orientalis DNA using primers 0611166 and 0611167.

The PCR reaction (50 μL) contained 100 ng of genomic I. orientalis DNA(preparable, e.g., using a MasterPure™ Yeast DNA Purification Kit fromEPICENTRE® Biotechnologies), 1× ThermoPol Reaction buffer (New EnglandBiolabs), 100 pmol each of primers 0611166 and 0611167, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 2 μL 100 mM MgSO₄, and 2 units of Vent_(R)®(exo-) DNA polymerase (New England Biolabs). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 94° C. for 2 minutes followed by 34 cycles each at 94° C. for 30seconds, 54° C. for 30 seconds, and 72° C. for 1 minute, with a finalextension at 72° C. for 10 minutes. Following thermocycling, the PCRreaction products were separated by 0.9% agarose gel electrophoresis inTAE buffer where an approximately 780 base pair PCR product was excisedfrom the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel, Bethlehem, Pa., USA) according to the manufacturer'sinstructions.

A PCR fragment containing the TAL terminator region and desiredadditional restriction sites and flanking DNA was amplified from pACN5(FIG. 19) using primers 0611168 and 0611169. The PCR reaction (50 μL)contained 1 μL of pACN5 mini-prep plasmid DNA, 1× iProof™ HF buffer(Bio-Rad Laboratories, Hercules, Calif., USA), 100 pmol each of primers0611168 and 0611169, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μLDMSO and 1 unit of iProof™ High Fidelity DNA polymerase (Bio-RadLaboratories). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 34 cycles each at 98° C. for 10 seconds, 59° C. for 20seconds, and 72° C. for 45 seconds, with a final extension at 72° C. for10 minutes. Following thermocycling, the PCR reaction products wereseparated by 0.9% agarose gel electrophoresis in TAE buffer where anapproximately 460 base pair PCR product was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

PCR then was used to create a single amplification product fusing bothof the products above. The PCR reaction (50 μL) contained 125 ng of thePDC promoter containing PCR product, 76 ng of the TAL terminatorcontaining PCR product, 1× ThermoPol Reaction buffer (New EnglandBiolabs), 100 pmol each of primers 0611166 and 0611169, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 2 μL 100 mM MgSO₄, and 2 units of Vent_(R)®(exo-) DNA polymerase (New England Biolabs). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 94° C. for 2 minutes followed by 34 cycles each at 94° C. for 30seconds, 54° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds,with a final extension at 72° C. for 10 minutes. Followingthermocycling, the PCR reaction products were separated by 0.9% agarosegel electrophoresis in TAE buffer where an approximately 1,250 base pairPCR product was excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions.

A PCR fragment containing the ENO1 promoter region and desiredadditional restriction sites and flanking DNA was amplified from pACN43(FIG. 22) using primers 0611170 and 0611171. The PCR reaction (50 μL)contained 1 μL of pACN43 mini-prep plasmid DNA, 1× Phusion HF buffer(New England Biolabs), 100 pmol each of primers 0611170 and 0611171, 200μM each of dATP, dCTP, dGTP, and dTTP, and 1 unit of Phusion™High-Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 30 seconds followed by 34 cycleseach at 98° C. for 10 seconds, 59° C. for 20 seconds, and 72° C. for 45seconds, with a final extension at 72° C. for 10 minutes. Followingthermocycling, the PCR reaction products were separated by 0.9% agarosegel electrophoresis in TAE buffer where an approximately 1050 base pairPCR product was excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions.

A PCR fragment containing the RKI terminator region followed by the URA3promoter region and the 5′ end of the URA3 ORF, along with desiredadditional restriction sites and flanking DNA, was amplified from pACN43(FIG. 22) using primers 0611172 and 0611173. The PCR reaction (50 μL)contained 1 μL of pACN43 mini-prep plasmid DNA, 1× iProof™ HF buffer(Bio-Rad Laboratories), 100 pmol each of primers 0611172 and 0611173,200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μL DMSO and 1 unit ofiProof™ High Fidelity DNA polymerase (Bio-Rad Laboratories). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 30 seconds followed by 34 cycleseach at 98° C. for 10 seconds, 59° C. for 20 seconds, and 72° C. for 45seconds, with a final extension at 72° C. for 10 minutes. Followingthermocycling, the PCR reaction products were separated by 0.9% agarosegel electrophoresis in TAE buffer where an approximately 1400 base pairPCR product was excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions.

PCR was used to create a single amplification product fusing both of theproducts above. The PCR reaction (50 μL) contained 93 ng of the ENO1promoter containing PCR product (supra); 125 ng of the RKI terminator,URA3 promoter and partial ORF containing PCR product (supra); 1× PhusionHF buffer (New England Biolabs); 100 pmol each of primers 0611170 and0611173; 200 μM each of dATP, dCTP, dGTP, and dTTP; 1.5 μL DMSO; and 1unit of Phusion™ High-Fidelity DNA polymerase (New England Biolabs). ThePCR was performed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 30 seconds followed by 34 cycleseach at 98° C. for 10 seconds, 56° C. for 20 seconds, and 72° C. for 2minutes and 45 seconds, with a final extension at 72° C. for 10 minutes.Following thermocycling, the PCR reaction products were separated by0.9% agarose gel electrophoresis in TAE buffer where an approximately2460 base pair PCR product was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

To create a recipient vector for the PCR products, the plasmid pMhCt017(the standard cloning vector pUC19 with an irrelevant insert) wasdigested with Hind III and EcoRI, treated with 10 units calf intestinalphosphatase (New England Biolabs) at 37° C. for 30 minutes, and purifiedby 0.9% agarose gel electrophoresis in TAE buffer, and an approximately2.6 kbp band was excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions. The resulting Hind III to EcoRI purified fragment wasidentical to that found in pUC18 (Yanisch-Perron, C., Vieira, J. andMessing, J. (1985) Gene, 33, 103-119).

The purified 1250 bp and 2460 bp PCR products from above were theninserted into the digested pMhCt017 fragment using an IN-FUSION™Advantage PCR Cloning Kit (Clontech) in a total reaction volume of 10 μLcomposed of 125 ng pMhCt017 Hind III to EcoRI vector fragment, 92 ng ofthe PDC promoter and TAL terminator PCR product, 165 ng of the ENO1promoter and URA3 promoter and partial ORF containing PCR product, 1×In-Fusion reaction buffer (Clontech) and 1 μL of IN-FUSION™ enzyme(Clontech). The reaction was incubated at 37° C. for 15 minutes, 50° C.for 15 minutes, and then placed on ice. The reaction then was dilutedwith 40 μL of TE buffer and 2.5 μL was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired PCRproducts by Apa LI digestion. A clone yielding the desired band sizeswas confirmed to be correct by DNA sequencing and designated pMhCt068.

The plasmid pMhCt068 contains the PDC promoter region followed by NheIand AscI restriction sites for addition of an ectopic gene of interestdescribed herein, the TAL terminator, the ENO1 promoter region followedby XbaI and PacI restriction sites for cloning of a second ectopic geneof interest described herein, the RKI terminator, the I. orientalis URA3promoter and the 5′ end of the I. orientalis URA3 ORF. Plasmid pMhCt068was found to have an A to T nucleotide change at about 200 bp into thePDC promoter, a G to T change at about ⅔ of the way into the PDCpromoter, and a premature start codon (ATG) present on the 5′ side ofthe NheI restriction site. Accordingly, a corrected version of pMhCt068was constructed as described below.

The PDC promoter region was PCR amplified from pACN5 (FIG. 19) withprimer 0611166 and 0611828, which do not introduce the undesired startcodon above. The PCR reaction (50 μL) contained 1 μL of a 1 to 50dilution of a mini-prep of pACN5, 1× ThermoPol Reaction buffer (NewEngland Biolabs), 100 pmol each of primers 0611166 and 0611828, 200 μMeach of dATP, dCTP, dGTP, and dTTP, 2 μL 100 mM MgSO₄, and 2 units ofVent_(R)® (exo-) DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 94° C. for 2 minutes followed by 34 cycleseach at 94° C. for 30 seconds, 54° C. for 30 seconds, and 72° C. for 1minute, with a final extension at 72° C. for 10 minutes. Followingthermocycling, the PCR reaction products were separated by 0.9% agarosegel electrophoresis in TAE buffer where an approximately 780 base pairPCR product was excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions.

The PDC promoter containing PCR product was then fused to the TALterminator containing PCR product as described above. Since the TALterminator PCR fragment was made with the 0611168 primer, the resultingPCR fusion products should be a mixture, with products that lack thepremature start codon and include the undesired start codon. Theresulting ˜1250 bp PCR product was purified and combined via IN-FUSION™Advantage PCR Cloning Kit (Clontech) with the the RKI terminator, URA3promoter and partial ORF containing fusion PCR product and pUC18 asdescribed above. A clone yielding the expected ApaLI digestion patternwas shown to be correct by DNA sequencing, including the desired absenceof mutations in the PDC promoter and lack of premature ATG 5′ of theNheI restriction site, and designated pMhCt082.

Construction of a Right-Hand Fragment

The empty vector right-hand construct, pMhCt069, was cloned in multiplesteps as described below.

A PCR fragment containing the 3′ end of the I. orientalis URA3 ORF, theURA3 terminator (the 275 bp downstream of the URA3 stop codon), the URA3promoter (to serve as a repeat region for looping out of the markerafter integration into the yeast host) and desired additionalrestriction sites and flanking DNA was amplified from pACN43 (FIG. 22)using primers 0611174 and 0611175. The PCR reaction (50 μL) contained 1μL of pACN43 mini-prep plasmid DNA, 1× Phusion HF buffer (New EnglandBiolabs), 100 pmol each of primers 0611174 and 0611175, 200 μM each ofdATP, dCTP, dGTP, and dTTP, and 1 unit of Phusion™ High-Fidelity DNApolymerase (New England Biolabs). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific) programmed for one cycle at 98° C.for 30 seconds followed by 34 cycles each at 98° C. for 10 seconds, 59°C. for 20 seconds, and 72° C. for 45 seconds, with a final extension at72° C. for 10 minutes. Following thermocycling, the PCR reactionproducts were separated by 0.9% agarose gel electrophoresis in TAEbuffer where an approximately 1210 bp PCR product was excised from thegel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions.

A PCR fragment containing the TDH3 promoter region and desiredadditional restriction sites and flanking DNA was amplified from pACN23(FIG. 20) using primers 0611176 and 0611177. The PCR reaction (50 μL)contained 1 μL of pACN23 mini-prep plasmid DNA, 1× Phusion HF buffer(New England Biolabs), 100 pmol each of primers 0611176 and 0611177, 200μM each of dATP, dCTP, dGTP, and dTTP, and 1 unit of Phusion™High-Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 30 seconds followed by 34 cycleseach at 98° C. for 10 seconds, 59° C. for 20 seconds, and 72° C. for 45seconds, with a final extension at 72° C. for 10 minutes. Followingthermocycling, the PCR reaction products were separated by 0.9% agarosegel electrophoresis in TAE buffer where an approximately 1028 bp PCRproduct was excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions.

A PCR fragment containing the region 3′ of the stop codon of the I.orientalis PDC gene region (PDC terminator region) and desiredadditional restriction sites and flanking DNA was amplified from I.orientalis genomic DNA using primers 0611178 and 0611179. The PCRreaction (50 μL) contained 100 ng of I. orientalis genomic DNA, 1×iProof™ HF buffer (Bio-Rad Laboratories), 100 pmol each of primers0611178 and 0611179, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μLDMSO and 1 unit of iProof™ High Fidelity DNA polymerase (Bio-RadLaboratories). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 34 cycles each at 98° C. for 10 seconds, 59° C. for 20seconds, and 72° C. for 45 seconds, with a final extension at 72° C. for10 minutes. Following thermocycling, the PCR reaction products wereseparated by 0.9% agarose gel electrophoresis in TAE buffer where anapproximately 938 base pair PCR product was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

PCR was used to create a single amplification product fusing both of thelast two PCR products described above. The PCR reaction (50 μL)contained 125 ng of the TDH3 promoter containing PCR product, 114 ng ofthe PDC terminator region containing PCR product, 1× Phusion HF buffer(New England Biolabs), 100 pmol each of primers 0611176 and 0611179, 200μM each of dATP, dCTP, dGTP, and dTTP, and 1 unit of Phusion™High-Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 30 seconds followed by 34 cycleseach at 98° C. for 10 seconds, 56° C. for 20 seconds, and 72° C. for 2minutes and 30 seconds, with a final extension at 72° C. for 10 minutes.Following thermocycling, the PCR reaction products were separated by0.9% agarose gel electrophoresis in TAE buffer where an approximately1966 base pair PCR product was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The purified 1210 bp PCR product and the 1966 bp PCR fusion product fromabove were then inserted into the Hind III and EcoRI digested pMhCt017fragment as described above using an IN-FUSION™ Advantage PCR CloningKit (Clontech) in a total reaction volume of 10 μL composed of 125 ngpMhCt017 Hind III to EcoRI vector fragment, 54 ng of PCR productcontaining the 3′ end of the URA3 ORF followed by the URA3 terminator,200 ng of the TDH3 promoter and PDC terminator fusion PCR product, 1×In-Fusion reaction buffer (Clontech) and 1 μL of IN-FUSION™ enzyme(Clontech). The reaction was incubated at 37° C. for 15 minutes, 50° C.for 15 minutes, and then placed on ice. The reaction was diluted with 40μL of TE buffer and 2.5 μL was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired PCRproducts by Apa LI digestion. A clone yielding the desired band sizeswas confirmed to be correct by DNA sequencing and designated pMhCt069.

Plasmid pMhCt069 contains the 3′ end of the I. orientalis URA3 marker,the corresponding URA3 terminator, the URA3 promoter (for later loopingout of the URA3 marker), the TDH3 promoter, XbaI and PacI restrictionsites for subcloning of desired genes for ectopic expression, and the 3′flanking region of the PDC locus.

Example 2F: Construction of Insertion Vector Fragments for ExpressingMultiple Exogenous Genes at the Adh9091 Locus

The following insertion vector fragments were designed using a similarapproach to that described in Example 2E in order to replace anendogenous I. orientalis adh9091 gene with a cassette that expressesmultiple genes of interest described herein.

Construction of a Left-Hand Fragment

An empty vector left-hand construct, pGREr125, was cloned in multiplesteps as described below.

A construct comprising the 5′ flank needed for homologous recombinationat the I. orientalis adh9091 locus and the empty expression cassette PDCpromoter/TAL terminator was PCR cloned into vector plasmid pCR2.1-TOPO(Invitrogen). The PDC promoter fragment was PCR amplified from plasmidpACN5 (FIG. 19) using primers 0611250 and 0611251. The PCR reaction (50μL) contained 15 ng of plasmid pACN5 DNA, 1× Phusion HF buffer (NewEngland Biolabs), 50 pmol each of primers 0611250 and 0611251, 200 μMeach of dATP, dCTP, dGTP, and dTTP, 1.5 μL of 100% DMSO (New EnglandBiolabs) and 1 unit of Phusion High Fidelity DNA polymerase (New EnglandBiolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 2 minutesfollowed by 35 cycles each at 98° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute and 30 seconds, with a final extensionat 72° C. for 7 minutes. Following thermocycling, the PCR reactionproducts were separated by 0.8% agarose gel electrophoresis in TBEbuffer where an approximately 900 bp PCR product was excised from thegel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen) accordingto the manufacturer's instructions. The total length of the resultingPCR fragment was approximately 753 bp with a NotI restriction site atthe 5′ end of the fragment and a PacI and an XbaI restriction site atthe 3′ end of the fragment.

A second PCR fragment containing 5′ homology to the PCR product aboveincluding the XbaI and PacI restriction sites was generated to amplifythe TAL terminator region from plasmid pACN5 (FIG. 19) using primers0611252 and 0611253. The PCR reaction (50 μL) contained 15 ng of plasmidpACN5 DNA (supra), 1× Phusion HF buffer (New England Biolabs), 50 pmoleach of primers 0611252 and 0611253, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1.5 μL of 100% DMSO (New England Biolabs) and 1 unit ofPhusion High Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 2 minutes followed by 35 cycleseach at 98° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute and 30 seconds, with a final extension at 72° C. for 7 minutes.Following thermocycling, the PCR reaction products were separated by0.8% agarose gel electrophoresis in TBE buffer where an approximately900 bp PCR product was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions. The total length of the resulting PCR fragment was about435 bp with XbaI and PacI restriction sites at the 5′ end of thefragment and a PmeI restriction site at the 3′ end.

The 753 bp fragment and the 435 bp fragment were fused together by PCRusing the primers 0611250 and 0611253, leading to a resulting 1149 bpfragment in which the PDC promoter is upstream of the TAL terminator.The PCR reaction (50 μL) contained 125 ng of the 753 bp fragment, 75 ngof the 435 bp fragment, 1× Phusion HF buffer (New England Biolabs), 50pmol each of primers 0611250 and 0611253, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs) and 1 unit ofPhusion High Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 2 minutes followed by 35 cycleseach at 98° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute, with a final extension at 72° C. for 7 minutes. Followingthermocycling, the PCR reaction product was separated by 0.8% agarosegel electrophoresis in TBE buffer where an approximately 1149 bp PCRproduct was excised from the gel and purified using a QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.

A PCR fragment containing 3′ homology to the 1149 bp PCR product aboveincluding the NotI restriction site, was generated to amplify the 5′flank for the I. orientalis adh9091 locus using primers 0611254 and0611255. The PCR reaction (50 μL) contained 15 ng of plasmid pHJJ27(FIG. 21) as template DNA, 1× Phusion HF buffer (New England Biolabs),50 pmol each of primers 0611254 and 0611255, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs) and 1 unit ofPhusion High Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 2 minutes followed by 35 cycleseach at 98° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute and 30 seconds, with a final extension at 72° C. for 7 minutes.Following thermocycling, the PCR reaction products were separated by0.8% agarose gel electrophoresis in TBE buffer where an approximately900 bp PCR product was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions. The total length of the resulting PCR fragment isapproximately 891 bp with an HpaI restriction site at the 5′ end of thefragment and a NotI restriction site at the 3′ end.

The 891 bp fragment then was fused upstream of the 1149 bp PDCpromoter/TAL terminator fragment by PCR using the primers 0611254 and0611253 generating an approximately 2005 bp fragment. The PCR reaction(50 μL) contained 125 ng of the 1149 bp fragment, 95 ng of the 891 bpfragment, 1× Phusion HF buffer (New England Biolabs), 50 pmol each ofprimers 0611254 and 0611253, 200 μM each of dATP, dCTP, dGTP, and dTTP,1.5 μL of 100% DMSO (New England Biolabs) and 1 unit of Phusion HighFidelity DNA polymerase (New England Biolabs). The PCR was performed inan EPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for onecycle at 98° C. for 2 minutes followed by 35 cycles each at 98° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, with afinal extension at 72° C. for 7 minutes. Following thermocycling, thePCR reaction products were separated by 0.8% agarose gel electrophoresisin TBE buffer where an approximately 2005 bp PCR product was excisedfrom the gel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen)according to the manufacturer's instructions.

The resulting 2005 bp fragment, comprising the 5′ flank for integrationat the adh9091 locus, the PDC promoter and the TAL terminator, wascloned into pCR2.1-TOPO vector and transformed into One-Shot TOP10 E.coli cells using a TOPO TA Cloning kit (Invitrogen) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired fragmentby Aval digestion. A done yielding the desired band sizes was confirmedand designated pGMEr112. Plasmid pGMEr112 comprises the 5′ flank forhomologous recombination at the adh9091 locus followed the emptyexpression cassette PDC promoter/TAL terminator.

The truncated 5′ URA3 marker gene driven by the URA3 promoter fragmentwas PCR amplified from plasmid pHJJ27 (FIG. 21) using primers 0611283and 0611263. The PCR reaction (50 μL) contained 15 ng of plasmid pHJJ27,1× Phusion HF buffer (New England Biolabs), 50 pmol each of primers0611263 and 0611283, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μLof 100% DMSO (New England Biolabs) and 1 unit of Phusion High FidelityDNA polymerase (New England Biolabs). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 98° C. for 2 minutes followed by 35 cycles each at 98° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds,with a final extension at 72° C. for 7 minutes. Following thermocycling,the PCR reaction products were separated by 0.8% agarose gelelectrophoresis in TBE buffer where an approximately 900 bp PCR productwas excised from the gel and purified using a QIAQUICK® Gel ExtractionKit (Qiagen) according to the manufacturer's instructions. The totallength of the resulting PCR fragment was approximately 885 bp with HpaIand PmeI restriction sites at the 5′ end of the fragment and a NheIrestriction site at its 3′ end.

The resulting 885 bp fragment was cloned into pCR2.1-TOPO vector andtransformed into One-Shot TOP10 E. coli cells using a TOPO TA Cloningkit (Invitrogen) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion of the desired insert by Bg/l digestion. A cloneyielding the desired band sizes was confirmed and designated pGMEr108.Plasmid pGMEr108 comprises the URA3 promoter followed by the truncated5′ segment of the URA3 gene, such fragment is flanked by HpaI and PmeIrestriction sites at the 5′ end and by the NotI restriction site at the3′ end.

A 1998 bp HpaI and PmeI restriction fragment from plasmid pGMEr112(supra), comprising the 5′ adh9091 flank followed by the construct PDCpromoter/TAL terminator, was ligated to the 4806 bp vector from pGMEr108(supra) linearized by HpaI and Pme I. The double restriction reactionproducts were separated by 0.8% agarose gel electrophoresis in TBEbuffer where the 1998 bp insert fragment and the 4806 bp vector fragmentwere excised from the gel and purified using a QIAQUICK® Gel ExtractionKit (Qiagen) according to the manufacturer's instructions. The ligationreaction was performed using a 1:3 vector insert ratio; in particularthe reaction was set up with 2 μL of the 4806 bp linearized vector, 6 μLof the 1998 bp insert fragment, 9 μL of 2× Quick Ligation reactionBuffer and 1 μL Quick T4 DNA Ligase (New England Biolabs), and performedaccording to the manufacturer's instructions.

Five μL of the ligation product was transformed into E. coli XL10-GoldUltracompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired insertby Hind III digestion. A clone yielding the desired band sizes wasconfirmed and designated pGMEr117.

Plasmid pGMEr117 comprises the 5′ adh9091 flank, followed by the emptyexpression cassette PDC promoter/TAL terminator and by the truncated 5′URA3 gene driven by the URA3 promoter. Additionally, plasmid pGMEr117bears two different XbaI restriction sites: a first restriction sitebetween the PDC promoter and the TAL terminator (and adjacent torestriction site Pac I) which can be used to insert the gene ofinterest, and a second XbaI restriction site that was inherited from theoriginal pCR2.1-TOPO back bone. In order to eliminate this second XbaIrestriction site, plasmid pGMEr117 was digested with restriction enzymeApaI, and the linearized plasmid was then treated with the enzyme DNApolymerase I, large (Klenow) fragment (New England Biolabs) according tothe manufacturer's instructions. The resulting linear vector (containingblunt ends) was digested with restriction enzyme Eco RV, which cut a 43bp fragment from the vector comprising the XbaI restriction site. Therestriction reaction products were separated by 0.8% agarose gelelectrophoresis in TBE buffer and the 6761 bp vector fragment wasexcised from the gel and purified using a QIAQUICK® Gel Extraction Kit(Qiagen) according to the manufacturer's instructions. The self ligationreaction was set up with 3 μL of the linearized vector, 6 μL of steriledouble-distilled water, 10 μL of 2× Quick Ligation reaction Buffer and 1μL Quick T4 DNA Ligase (New England Biolabs) and performed according tothe manufacturer's instructions.

Five μL of the ligation product was transformed into E. coli XL10-GoldUltracompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired insertby XbaI digestion. A clone yielding the desired band sizes was confirmedand designated pGMEr122.

The ENO1 promoter fragment was PCR amplified from plasmid pACN43 (FIG.22) using the primers 0611295 and 0611296. The PCR reaction (50 μL)contained 15 ng of plasmid pACN43, 1× Phusion HF buffer (New EnglandBiolabs), 50 pmol each of primers 0611295 and 0611296, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs)and 1 unit of Phusion High Fidelity DNA polymerase (New EnglandBiolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 2 minutesfollowed by 35 cycles each at 98° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute, with a final extension at 72° C. for 7minutes. Following thermocycling, the PCR reaction products wereseparated by 0.8% agarose gel electrophoresis in TBE buffer where anapproximately 1009 bp PCR product was excised from the gel and purifiedusing a QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions. The total length of the resulting PCRfragment was approximately 1009 bp with a PmeI restriction site at the5′ end of the fragment and ApaI and NruI restriction sites at the 3′end.

A second PCR fragment containing 5′ homology to the PCR product above,including the NruI and the ApaI restriction sites, was generated toamplify the RKI terminator region from plasmid pACN43 (FIG. 22) usingthe primers 0611297 and 0611298. The PCR reaction (50 μL) contained 15ng of plasmid pACN43 DNA, 1× Phusion HF buffer (New England Biolabs), 50pmol each of primers 0611297 and 0611298, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs) and 1 unit ofPhusion High Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 2 minutes followed by 35 cycleseach at 98° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute, with a final extension at 72° C. for 7 minutes. Followingthermocycling, the PCR reaction products were separated by 0.8% agarosegel electrophoresis in TBE buffer where an approximately 438 bp PCRproduct was excised from the gel and purified using a QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.The total length of the resulting PCR fragment was about 438 bp withNruI and ApaI restriction sites at the 5′ end of the fragment and a PmeIrestriction site at the 3′ end of the fragment.

The 1009 bp promoter fragment and the 438 bp terminator fragment werefused together by PCR using primers 0611295 and 0611298, leading to anapproximately 1447 bp fragment in which the ENO1 promoter is upstream ofthe RKI terminator. The PCR reaction (50 μL) contained 125 ng of the1009 bp fragment, 65 ng of the 438 bp fragment, 1× Phusion HF buffer(New England Biolabs), 50 pmol each of primers 0611295 and 0611298, 200μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μL DMSO (New England Biolabs)and 1 unit of Phusion High Fidelity DNA polymerase (New EnglandBiolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 2 minutesfollowed by 35 cycles each at 98° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 2 minute, with a final extension at 72° C. for 7minutes. Following thermocycling, the PCR reaction product was separatedby 0.8% agarose gel electrophoresis in TBE buffer where an approximately1447 bp PCR product was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions.

The resulting 1447 bp fragment, comprising the ENO1 promoter/RKIterminator construct, was cloned into pCR2.1-TOPO vector and transformedinto One-Shot TOP10 E. coli cells using a TOPO TA Cloning kit(Invitrogen) according to the manufacturer's instructions. Transformantswere plated onto 2× YT+amp plates and incubated at 37° C. overnight.Several of the resulting transformants were screened for properinsertion of the desired fragment by Bam HI digestion. A clone yieldingthe desired band sizes was confirmed and designated pGMEr114, comprisingthe empty expression cassette ENO1 promoter/RKI terminator.

Plasmids pGMEr122 and pGMEr114 were digested with restriction enzymePmeI at 37° C. for 3 hours. Approximately one hour before stopping eachdigestion reaction, 1 μL of Calf Intestinal Alkaline Phosphatase (NewEngland Biolabs) was added to each digestion tube in order tode-phosphorylate the ends and prevent self-ligation. The resulting 6761bp vector fragment from plasmid pGMEr122, and the resulting insertfragment comprising the construct ENO1 promoter/TAL terminator (1439 bp)from plasmid pGMEr114, were separated by 0.8% agarose gelelectrophoresis in 1×TBE buffer, excised from the gel, and purifiedusing the QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions.

A subsequent ligation reaction was then prepared comprising 3 μL of thevector fragment from plasmid pGMEr122, 4 μL of the insert fragment fromplasmid pGMEr114, 2 μL of sterile dd water, 10 μL of 2× Quick LigaseBuffer and 1 μL of Quick T4 Ligase (New England Biolabs) and performedaccording to the manufacturer's instructions. A 5 μL aliquot of theligation reaction above was transformed into XL10-Gold® UltracompetentE. coli cells (Agilent Technologies) according to the manufacturer'sinstructions. Transformants were plated onto 2× YT+amp plates andincubated at 37° C. overnight. Several of the resulting transformantswere screened for proper insertion of the desired insert by digestionusing XbaI and Bst BI. A clone yielding the desired band sizes wasconfirmed by sequencing and designated pGMEr125. The ENO1 promoter/RKIterminator construct was inserted in opposite orientations, resulting intwo versions of plasmid pGMEr125 designated (a) and (b) (FIGS. 4 and 5).

The plasmids pGMEr125a and pGMEr125b contain the PDC promoter region,the TAL terminator, the ENO1 promoter region, the RKI terminator, the I.orientalis URA3 promoter, the 5′ end of the corresponding URA3 markerand the 5′ flanking region of the I. orientalis adh9091 locus.

Construction of a Right-Hand Fragment

An empty vector right-hand construct, pGREr121, was cloned in multiplesteps as described below.

The TDH3 promoter fragment was PCR amplified from plasmid pACN23 (FIG.20) using primers 0611256 and 0611257. The PCR reaction (50 μL)contained 15 ng of plasmid pACN23 DNA, 1× Phusion HF buffer (New EnglandBiolabs), 50 pmol each of primers 0611256 and 0611257, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs)and 1 unit of Phusion High Fidelity DNA polymerase (New EnglandBiolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 2 minutesfollowed by 35 cycles each at 98° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute and 30 seconds, with a final extensionat 72° C. for 7 minutes. Following thermocycling, the PCR reactionproducts were separated by 0.8% agarose gel electrophoresis in TBEbuffer where an approximately 994 bp PCR product was excised from thegel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen) accordingto the manufacturer's instructions. The total length of the resultingPCR fragment was approximately 994 bp with a Sfol restriction site atthe 5′ end of the fragment and PacI and NruI restriction sites at the 3′end.

A second PCR fragment containing 5′ homology to the PCR product above,including the NruI and PacI restriction sites, was generated to amplifythe TKL terminator region from plasmid pACN23 (FIG. 20) using primers0611258 and 0611259. The PCR reaction (50 μL) contained 15 ng of plasmidpACN23 DNA, 1× Phusion HF buffer (New England Biolabs), 50 pmol each ofprimers 0611258 and 0611259, 200 μM each of dATP, dCTP, dGTP, and dTTP,1.5 μL of 100% DMSO (New England Biolabs) and 1 unit of Phusion HighFidelity DNA polymerase (New England Biolabs). The PCR was performed inan EPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for onecycle at 98° C. for 2 minutes followed by 35 cycles each at 98° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30seconds, with a final extension at 72° C. for 7 minutes. Followingthermocycling, the PCR reaction products were separated by 0.8% agarosegel electrophoresis in TBE buffer where an approximately 469 bp PCRproduct was excised from the gel and purified using a QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.The total length of the resulting PCR fragment was about 469 bp withNruI and PacI restriction sites at the 5′ end of the fragment and a NotIrestriction site at the 3′ end.

The 994 bp and 469 bp fragments above were fused together by PCR usingprimers 0611256 and 0611259, leading to an approximately 1433 bpfragment in which the TDH3 promoter is upstream of the TKL terminator.The PCR reaction (50 μL) contained 125 ng of the 994 bp fragment, 60 ngof the 469 bp fragment, 1× Phusion HF buffer (New England Biolabs), 50pmol each of primers 061159 and 0611256, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs) and 1 unit ofPhusion High Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 2 minutes followed by 35 cycleseach at 98° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute, with a final extension at 72° C. for 7 minutes. Followingthermocycling, the PCR reaction product was separated by 0.8% agarosegel electrophoresis in TBE buffer where an approximately 1433 bp PCRproduct was excised from the gel and purified using a QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.

A PCR fragment containing 5′ homology to the 3′ end of the 1433 bp PCRproduct above which includes the NotI restriction site was generated toamplify the 3′ flank for the adh9091 locus using primers 0611260 and0611261. The PCR reaction (50 μL) contained 15 ng of plasmid pHJJ27 DNA(FIG. 21) as template DNA, 1× Phusion HF buffer (New England Biolabs),50 pmol each of primers 0611260 and 0611261, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1.5 μL of 100% DMSO (New England Biolabs) and 1 unit ofPhusion High Fidelity DNA polymerase (New England Biolabs). The PCR wasperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 2 minutes followed by 35 cycleseach at 98° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute and 30 seconds, with a final extension at 72° C. for 7 minutes.Following thermocycling, the PCR reaction products were separated by0.8% agarose gel electrophoresis in TBE buffer where an approximately1019 bp PCR product was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions. The total length of the resulting PCR fragment isapproximately 1019 bp with a NotI restriction site at the 5′ end of thefragment and an ApaI restriction site at the 3′ end.

The 1019 bp fragment was then fused downstream of the 1433 bp TDH3promoter/TKL terminator fragment by PCR using primers 0611256 and0611261 generating an approximately 2405 bp fragment. The PCR reaction(50 μL) contained 125 ng of the 1433 bp fragment, 90 ng of the 1019 bpfragment, 1× Phusion HF buffer (New England Biolabs), 50 pmol each ofprimers 0611256 and 0611261, 200 μM each of dATP, dCTP, dGTP, and dTTP,1.5 μL of 100% DMSO (New England Biolabs) and 1 unit of Phusion HighFidelity DNA polymerase (New England Biolabs). The PCR was performed inan EPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for onecycle at 98° C. for 2 minutes followed by 35 cycles each at 98° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, with afinal extension at 72° C. for 7 minutes. Following thermocycling, thePCR reaction products were separated by 0.8% agarose gel electrophoresisin TBE buffer where an approximately 2405 bp PCR product was excisedfrom the gel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen)according to the manufacturer's instructions.

The resulting 2405 bp fragment comprising the 3′ flank for integrationat the adh9091 locus downstream of the TDH3 promoter/TAL terminatorconstruct was cloned into pCR2.1-TOPO vector and transformed intoOne-Shot TOP10 E. coli cells using a TOPO TA Cloning kit, (Invitrogen)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion of thedesired fragment by Aval digestion. A clone yielding the desired bandsizes was confirmed and designated pGMEr113. Plasmid pGMEr113 comprisesthe 3′ flank for homologous recombination at the I. orientalis adh9091locus preceded by the empty expression cassette TDH3 promoter/TKLterminator.

PCR was used to amplify the truncated 3′ fragment of the URA3 ORF, theURA3 terminator, and the URA3 promoter (to serve as a repeat region forlooping out of the marker after integration into the yeast host asdescribed above) from plasmid pHJJ27 (FIG. 21) using primers 0611264 and0611284. The PCR reaction (50 μL) contained 15 ng of plasmid pHJJ27 DNA,1× Phusion HF buffer (New England Biolabs), 50 pmol each of primers0611264 and 0611284, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μLof 100% DMSO (New England Biolabs) and 1 unit of Phusion High FidelityDNA polymerase (New England Biolabs). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 98° C. for 2 minutes followed by 35 cycles each at 98° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 1 minute and 30 seconds,with a final extension at 72° C. for 7 minutes. Following thermocycling,the PCR reaction products were separated by 0.8% agarose gelelectrophoresis in TBE buffer where an approximately 1324 bp PCR productwas excised from the gel and purified using a QIAQUICK® Gel ExtractionKit (Qiagen) according to the manufacturer's instructions. The totallength of the resulting PCR fragment was approximately 1324 bp with aNheI restriction site at the 5′ end of the fragment and ApaI and Sfolrestriction sites at the 3′ end.

The gel-purified 1324 bp fragment above was cloned into the pCR2.1-TOPOvector and transformed into One-Shot TOP10 E. coli cells using a TOPO TACloning kit (Invitrogen) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion of the desired insert by Hind III digestion. A cloneyielding the desired band sizes was confirmed and designated pGMEr109.Plasmid pGMEr109 comprises the 3′ fragment of the URA3 ORF and the URA3terminator, followed by the URA3 promoter. The upstream portion of the3′ fragment of the URA3 gene in plasmid pGMEr109 bears a 460 bp homologywith the extremity of the truncated 5′ URA3 fragment cloned into plasmidpGMEr108. The region of homology allows recombination between the twoportions of the gene creating a functional selection marker uponco-transformation of the host organism with the construct containingboth segments.

Plasmid pGMEr109 was digested with KpnI, and treated with DNA polymeraseI, large (Klenow) fragment (New England Biolabs) according to themanufacturer's instructions. The linearized pGMEr109 plasmid (containingblunt ends) was digested with Bam HI. The products were separated by0.8% agarose gel electrophoresis in TBE buffer and the 5247 bp vectorfragment was excised from the gel and purified using a QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.

Plasmid pGMEr113 was digested with Bam HI and Eco RV resulting in a 2466bp fragment bearing the construct TDH3 promoter/TKL terminator followedby the truncated 3′ fragment of the URA3 ORF with the URA3 terminator,followed by the URA3 promoter. The double restriction reaction productswere separated by 0.8% agarose gel electrophoresis in TBE buffer and theapproximately 2466 bp vector fragment was excised from the gel andpurified using a QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions. The 2466 bp Bam HI/Eco RV digested fragmentthen was ligated to the 5247 bp vector fragment from plasmid pGMEr109.The ligation reaction was set up with 3 μL of the 5247 bp linearizedvector, 3 μL of the 2466 bp insert fragment, 3 μL of sterile dd water,10 μL of 2× Quick Ligation reaction Buffer and 1 μL Quick T4 DNA Ligase(New England Biolabs), and performed according to the manufacturer'sinstructions.

Five μL of the ligation product was transformed into E. coli XL10-GoldUltracompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired insertby digestion with XbaI and Pac I. A clone yielding the desired bandsizes was confirmed and designated pGMEr121 (FIG. 6).

Plasmid pGMEr121 contains the 3′ end of the I. orientalis URA3 markerfollowed by the corresponding URA3 promoter, the TDH3 promoter, the TKLterminator and the 3′ flanking region of the adh9091 locus.

Example 2G: Construction of I. Orientalis CNB1

I. orientalis CNB1 was constructed from I. orientalis CD1822 asdescribed below (see Example 1A for generation of I. orientalis CD1822from I. orientalis ATCC PTA-6658). Both copies of the URA3 genecontained in strain CD1822 were deleted to allow use of this gene as aselection marker for genetic engineering. URA3 is a versatile marker foryeast genetics due to the selection available for both the presence (bygrowth in uracil deficient media) and absence (by growth in the presenceof 5-fluoorotic acid) of the gene. Disruption of one of the URA3 geneswas done by replacement with a selection cassette containing the MEL5selection marker flanked by repeated DNA sequences. Strains testingpositive for the MEL5 selection cassette were then screened for the lossof MEL5 marker gene. Loss of the second URA3 gene was then selected forby growth in the presence of 5-fluoorotic acid.

CD1822 was transformed with 2.8 μg of Sac I/PspOMI digested DNA ofvector pMI458 (FIG. 27). Plasmid pMI458 contains the S. cerevisiae MEL5gene (SEQ ID NO: 255) under control of the I. orientalis PGK promoter(P-IoPGK, SEQ ID NO: 247), flanked by DNA fragments homologous tosequence upstream (P-IoURA3, SEQ ID NO: 253) and downstream (T-IoURA3,SEQ ID NO: 254) of the I. orientalis URA3 gene. The P-IoURA3 andT-IoURA3 fragments are in the same relative orientation as in the I.orientalis genome. Roughly 500 Mel+ colonies were obtained after fivedays at 30° C. Ten colonies were single colony isolated by inoculating a10 mL BFP (Butterfields Phosphate buffer) tube and plating 25 μL ontoDM1 X-α-gal plates. A single blue colony from each of the initialisolates was then picked onto YPD for further analysis.

PCR was used to screen transformants for the desired genetic events. Toobtain genomic DNA for use as template in PCR screenings, cells from 1.5mL overnight cultures were spun down in a screw-cap microcentrifuge tubeand resuspended in 0.2 ml of 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris-HCl pH 8.0, 1 mM Na₂EDTA pH 8.0 solution. 0.2 mL of aphenol:chloroform:isoamyl alcohol (25:24:1) mixture equilibrated with 10mM Tris pH 8.0/1 mM EDTA (Sigma) and 0.3 g of glass beads were added.The tube was shaken for 2 minutes at full speed with a Mini-BeadBeater-8(BioSpec). 0.2 mL of TE was added and the tube was vortexed briefly. Theaqueous phase was separated by centrifugation at 16,100×g for 5 minutes.The supernatant was removed to a new tube and 1 mL of 100% ethanoladded. The tube was placed at 20° C. for 30 minutes, centrifuged at16,100×g for 5 minutes, and the liquid decanted off. The DNA was airdried and resuspended in 500 μL TE.

The PCR screen for the desired 5′ cross-over was done using primersoCA405 and oCA406, which produce a 1.5 kbp product. The screen for thedesired 3′ cross-over was done using primers WG26 and CM647, whichproduce a 1.6 kbp product. Primers outside (farther upstream ordownstream) the URA3 regions used to create pMI458 are oCA405 and CM647,which produce a 3.2 kbp product for the wild type, 5.0 kbp product for apMI458 disrupted allele, and 2.2 kbp product when the selection markerhas been looped out. These PCR reactions were done using a 55° C.annealing temperature. PCR was also used to screen for the loss of URA3open reading frame using a four-primer approach. Primers pJLJ28 andpJLJ29 amplify an 800 bp fragment of the actin gene and primers pJLJ30and pJLJ31 amplify a 600 bp fragment of the URA3 gene. Use of all theprimers in one reaction provides a positive internal control (the actinfragment). Taq DNA polymerase from Roche was used as per manufacturer'sprotocol, with an annealing temperature of 61° C. Strains 1822ura hetMEL-1 and 1822ura het MEL-2 were confirmed as having integrated the MEL5selection cassette in the URA3 locus.

The MEL5 marker was then removed from the genomes of 1822ura het MEL-1and 1822ura het MEL-2 by allowing recombination between the KtSEQ1a (SEQID NO: 256) and KtSEQ1b (SEQ ID NO: 257) sequences. The MEL+ strainswere grown overnight in YPD media to an OD₆₀₀ of roughly 0.5 to 2.0. Thecultures were then diluted back to an OD₀₀ of approximately 0.00001 inYPD medium. 200 μL of culture dilution was transferred into each well ofa 96 well microtiter plate. The plates were covered with an adhesivecover and incubated in a 30° C. incubator, with maximum agitation for6-7 hours (roughly 6 cell divisions, depending on growth rate of thestrain). 100 μL from each well (approximately ˜1000 cfu per plate) wasplated onto DM1+X-α-gal medium. Plates were incubated at 30° C.overnight or at room temperature for 2 days to observe colordifferentiation. White colonies (putative mel-) were streaked ontosimilar media, and screened by PCR as described above. Two independentloop-outs were found, one from 1822ura het MEL-1, saved as 1822ura hetmel-1 and the other from 1822ura het MEL-2, saved as 1822ura het mel-2.Oddly, the vast majority of white colonies obtained did not give theexpected band of 2.2 kbp.

To obtain ura-derivatives, 1822ura het mel-1 and 1822ura het mel-2 weregrown overnight in YP5D media (YP+100 g/L Dextrose) and aliquots (0.5, 5and 50 μL) of the overnight culture were plated on ScD-2×FOA plates.FOA-resistant colonies were streaked for single colonies and verifiedfor the ura-phenotype by plating on ScD-ura plates. Two colonies from1822ura het mel-2 and six colonies from 1822ura het mel-1 were pickedfor further analysis. These colonies were grown overnight in YPD andgenomic DNA was extracted using the above phenol/chloroform method.

The presence of the URA3 open reading frame was screened with PCR; noneof the eight strains contained the URA3 gene. Two ura-descendents of1822ura het mel-1 were named yJLJ3 (CNB1) and yJLJ4. Based genomicsequencing, yJLJ3 (CNB1) and yJLJ4 were determined to contain a deletionof both the URA3 gene and a nearby permease gene; preferably only theURA3 gene would be deleted. To create a ura3 auxotroph of CD1822 withoutdisruption of this permease gene, CD1822 was transformed with 1 pg ofSac I/ApaI digested pCM208 (FIG. 28). Plasmid pCM208 contains DNAsequence homologous to the upstream (5′ URA flank (near), SEQ ID NO:258) and downstream (3′ URA3 flank (near), SEQ ID NO: 259) flankingregions of the I. orientalis URA3 gene. Roughly 200 Mel+ colonies wereobtained after five days at 30° C. Eight blue colonies were isolated bystreaking on ScD X-α-gal plates. PCR was used to screen transformantsfor the desired genetic events. The 5′ cross-over screen was done usingprimers oJY11 and oJY12, which produce a 0.9 kbp product in desiredtransformants. The 3′ cross-over screen was done using primers oJY13 andoJY14, which produce a 1.0 kbp product in desired transformants. Threeof eight colonies showed the desired PCR products. The MEL markers forthese colonies can be looped out and the second URA3 gene deleted asdescribed above. Alternatively, the URA3 and permease gene deletions instrains derived from yJLJ3 or yJLJ4 can be fixed in a one-steptransformation, as described in Example 2H.

Example 2H: Construction of MBin500 Control Strain Containing the URA3Selection Marker

As described supra, the I. orientalis strain designated CNB1 used hereinwas a uridine auxotroph due to the homozygous deletion of the URA3 gene.A heterozygous repair of the URA3 locus was made using the ura fixvector, pJLJ62 (FIG. 26) which contains a ura fix cassette comprised ofthe URA3 gene with 691 bp of 5′ flanking DNA, and 1500 bp of 3′ flankingDNA. The ura fix cassette is flanked by a 5′ NotI restriction site and a3′ ApaI restriction site. A restriction digest using NotI and ApaI wasperformed to remove the 2796 bp ura fix cassette from the vectorbackbone. The digest was purified using a QIAquick PCR Purification kit(Qiagen) as specified by the manufacturer. The DNA was eluted in glassdistilled water and 1 pg was used to transform I. orientalis CNB1.Transformants were selected on ura selection plates, and a single colonythat does not require uridine supplementation was designated MBin500.

Example 21: Removal of the URA3 Selection Marker

In order to isolate strains in which the URA3 selection marker gene wasremoved via recombination of the two URA3 promoter regions present inthe integration cassettes, the ura+ strain of interest was inoculatedinto in 3 mL of YP+10% glucose media and grown with shaking at 250 rpmat 37° C. for at least four hours and up to overnight. 50-100 μL of theculture was plated onto ScD FOA plates and grown at 37° C. for 48-60hours until colonies appeared. Growth on FOA selected for the removal ofthe URA3 marker since FOA is converted to a toxic compound by the URA3protein, resulting in the death of ura+ cells. Several FOA-resistantcolonies were purified twice by growing on YPD plates 37° C. Thesepurified isolates were then screened for appropriate URA3 loop-out viaPCR as described herein.

Example 2J: Procedure for Shake Flask Growth of Modified Yeast Strainsfor Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)Analysis and Enzyme Assays

Four mL of ura selection media was added to a 14 mL Falcon tube and thedesired strain was inoculated into this media using a sterile loop. Theculture was grown with shaking at 250 rpm overnight (˜16 hrs) at 37° C.For strains that have at least one wild-type copy of the I. orientalislocus, 500 μL of the overnight culture was added to a 125 mL baffledflask containing 25 mL of YP+10% glucose media. For pdcΔ/pdcΔ strains, 1mL of the overnight culture was added to a 125 mL baffled flaskcontaining 25 mL of liquid YP+100 g/L dextrose media. The flask wasgrown with shaking at 250 rpm at 37° C. SmaIl aliquots of the culturewere withdrawn at approximately hourly intervals and the OD₆₀₀ wasmeasured. The culture was grown until the OD₆₀₀ was between 4 and 6.

In order to prepare a small sample of cells for SDS-PAGE analysis, avolume of culture corresponding to 2.5 OD units was taken for theculture and placed in a 1.5 mL tube. The cells were pelleted at16,100×g, the supernatant removed, and the cell pellet stored at −20° C.until use.

The remaining cells in the growth flask were harvested by centrifugationat 2279×g at room temperature, the pellet was resuspended in 12.5 mL0.85 M NaCl, then centrifuged at 2279×g at room temperature. The pelletwas resuspended in 1 mL 0.85 M NaCl, and the resuspended cells weretransferred to a 2.0 mL tube and then pelleted at 16,100×g. Thesupernatant was then removed and the pellet stored at −20° C. if theywould be used for enzymatic assays within one week, or at −80° C. forlonger term storage.

For SDS-PAGE analysis of the cell pellet corresponding to 2.5 OD units,the cells were resuspended in 100 dH₂O, then 100 μL 0.2 M NaOH wasadded. The sample was incubated at room temperature for 5 minutes, thenthe cells were pelleted by centrifugation at 16,100×g and resuspended in100 μL SDS sample buffer (Bio-Rad Laboratories). The sample was heatedat 95° C. for 5 minutes and cells were pelleted by brief centrifugation.1 to 5 μL of the supernatant was analyzed on a Criterion 8-16% Pre-Castgel (Bio-Rad Laboratories) according to the manufacturer's instructions.Bands were visualized using InstantBlue™ Coomassie-Based StainingSolution (Expedeon Protein Solutions, San Diego, Calif., USA).

Example 2K: Procedure for Shake Flask Growth of Modified Yeast Strainsfor Product Analysis

Strains were streaked out for single colonies on Ura Selection Platesand incubated at 30° C. for 1-2 days. Seed cultures were prepared in 250ml baffled flasks containing 50 mL CNB1 shake flask media inoculatedwith 1-2 colonies from the Ura Selection Plate. Seed cultures were grownfor approximately 18 hours at 30° C. with shaking at 200 rpm. SmaIlaliquots of the culture were then withdrawn to measure the OD₆₀₀ untilreaching an OD₆₀₀ of 4-6. The residual glucose present was measuredusing an Uristix® Reagent Strip (Bayer, Elkhart, Ind., USA). The seedflask cultivation was used to inoculate 125 ml baffled flasks containing50 mL CNB1 shake flask media to an OD₆₀₀=0.2. Cultures were incubated at30° C. with shaking at 140 rpm for 20 hr. Samples of the broth wereremoved for analysis as described below. An aliquot of the sample wasused to measure the optical density (OD) of the culture and residualglucose present was measured using a Uristix® Reagent Strip. The rest ofthe sample was then centrifuged and the supernatant used for productanalysis.

Example 2L: Procedure for Fermentation of Modified Yeast Strains forProduct Analysis

Strains described herein are cultivated using a seed propagation stageand followed by a single stage fermentation in a 2 L bioreactor(Applikon, Foster City, Calif., USA).

For seed stage preparation 25 mL of 1× DM2 medium (adjusted to thedesired pH with KOH) was added to a 125 mL baffled flask, followed byinoculation with the strain of interest using a sterile loop. Theculture was grown with shaking at 250 rpm at the desired temperatureovernight for approximately 16 hr. SmaIl aliquots of the culture werethen withdrawn at approximately hourly intervals to measure the OD₆₀₀until reaching an OD₆₀₀ of 4-6.

The residual glucose present was measured using a Uristix® Reagent Strip(Bayer, Elkhart, Ind., USA). 12 mL of the culture was then added to 4 mLof sterile chilled 75% glycerol, mix thoroughly, and incubated on icefor ten minutes. The culture and glycerol mixture was then remixed and1.0 mL was aliquoted to each of 10 sterile 1.8 mL cryovials (ThermoScientific, Rochester, N.Y., USA) and stored at −80° C.

25 mL of the seed flasks cultivation was used to inoculate the 2 Lbioreactor containing 1.5 L of DM2 medium. The fermentation in thebioreactor was performed at a temperature of about 30° C.-40° C., withthe pH controlled in the range of about 2.0-7.0 and under agitation andaeration conditions that lead to an oxygen uptake rate (OUR) in therange of 2-45 mmol/hr. In the examples presented herein, thetemperature, pH and OUR for the culture in the bioreactor were 30° C.,4.0 and 25-30, respectively.

Samples of the fermentation broth were removed periodically foranalysis. Briefly, an aliquot of the sample was used to measure theoptical density (OD) of the culture, the glucose concentration and pH.The rest of the sample was then centrifuged. The pellet was stored at−80° C. for enzyme assays, and the supernatant was used for analysis of3-HP and other extracellular compounds All 3-HP production valuesreported herein are for the 48-hour time point in the fermentationunless specified otherwise. Carbon dioxide production and oxygenconsumption during the fermentation process were measured by determiningthe carbon dioxide content and oxygen content of the gasses vented fromthe bioreactor.

Example 2M: Procedure for Analysis of 3-HP and R-Alanine Produced byModified Yeast Strains

Culture samples were acidified by 10× dilution into 1% formic acid andfiltered through a 0.46 μm 96-well filter plate. Further dilution wasmade in water depending on analyte concentration in the sample. Afurther 10× dilution was made in a sample buffer of 1 mM NH₄Ac, 0.1% NH₃and 5 mg/L of ¹³C uniformly labeled 3-HP (as internal standard for3-HP), or 1% formic acid and 3 mg/L of ¹³C uniformly labeled β-alanine(as internal standard for 3-alanine). The total dilution factor wasapproximately 100 to 1000 was used depending on the concentrations ofβ-alanine or 3-HP.

A 2 μL sample was injected into an Agilent 1200 HPLC (Agilent)controlled by MassHunter program with an Agilent 6410 Triple Quad MS/MSdetector using the instrument settings and columns listed in Table 6.The ratio of the quantifying ion fragment peak area to its stableisotope counterpart (from internal standard) was used for quantificationto eliminate ion suppression effect and instrument drifting. Standarddeviation was below 5% from day to day assays.

TABLE 6 LC/MS/MS Settings for β-Alanine and 3-HP analysis β-Alanine 3-HP(¹³C 3-HP) (¹³C β-Alanine) Column Xbridge HILIC Silica Atlantis HILICSilica 3.5 μm, 2.1 × 150 mm 3 μm 2.1 × 150 mm Elution buffer 62%acetonitrile, 38% acetonitrile, 0.6% 0.35 mM NH₄Ac formic acid Flow rate(mL/min) 0.30 0.30 Column temperature 50° C. 45° C. Retention time (min)1.07 1.64 Run time (min) 3 3 Pre-cursor ion 89 (92) 90 (93) Product ionas 59 (61) 72 (75) quantifier Product ion as 41 (43) 30 (31) qualifierFragmentor Voltage 50 70 Collision energy 5 for quantifier; 21 for 3 forquantifier; qualifier 7 for qualifier Polarity Negative PositiveNebulizer N₂ 10 11 pressure(psi) N₂ flow (L/min) 32 35 N₂ temperature300° C. 340° C. Capillary (V) 4000 4000 Delta EMV 450 400

Example 3: Modified Yeast Strains Expressing 3-HP Fermentation PathwayGenes

One or more genes encoding enzymes involved in various 3-HP fermentationpathways can be expressed, either alone or in combination, in yeast hostcells. The 3-HP pathway enzymes may be expressed from exogenous genes,endogenous genes, or some combination thereof. Exogenous genes to beexpressed may be introduced into the yeast cell using gene expressionconstructs, e.g., expression constructs described in Example 2.Exogenous genes may be integrated into the host yeast genome at a singlesite or at multiple locations, and integration of the exogenous gene maybe coupled with deletion or disruption of a target gene at the insertionsite as described below.

Example 3A: Modified Yeast Strains Expressing Aspartate/MalonateSemialdehyde Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes PEP and/orpyruvate, OAA, aspartate, β-alanine, and malonate semialdehydeintermediates can be engineered by expressing one or more enzymesinvolved in the pathway. The expressed genes may include one or more ofa PPC, PYC, AAT, ADC, BAAT, gabT, 3-HPDH (including malonatesemialdehyde reductase), HIBADH, or 4-hydroxybutyrate dehydrogenasegene.

The expressed genes may be derived from a gene that is native to thehost cell. For example, where the yeast host cell is I. orientalis,expressed genes may be derived from an I. orientalis PYC (e.g., I.orientalis PYC gene encoding the amino acid sequence set forth in SEQ IDNO: 2 and/or comprising the coding region of the nucleotide sequence setforth in SEQ ID NO: 1), AAT (e.g., I. orientalis AAT gene encoding theamino acid sequence set forth in SEQ ID NO: 14 and/or comprising thecoding region of the nucleotide sequence set forth in SEQ ID NO: 13),BAAT (e.g., I. orientalis pyd4 homolog gene encoding the amino acidsequence set forth in SEQ ID NO: 20 and/or comprising the coding regionof the nucleotide sequence set forth in SEQ ID NO: 19), or 3-HPDH (e.g.,I. orientalis homolog to the YMR226C gene encoding the amino acidsequence set forth in SEQ ID NO: 26 and/or comprising the coding regionof the nucleotide sequence set forth in SEQ ID NO: 25) gene. Where theyeast host cell is another 3-HP tolerant yeast strain, gene sequencescan be obtained using techniques known in the art and the nativehomologs for pathways genes can be expressed exogenously or inconjunction with exogenous regulatory elements. Native pathway genes mayinclude one or more PPC, PYC, AAT, BAAT, and/or 3-HPDH genes.

Alternatively, one or more of the expressed 3-HP genes may be derivedfrom a source gene that is non-native to the host cell. For example,where the yeast host cell is I. orientalis, the cell may be engineeredto express one or more non-native PYC genes such as an R. sphaeroidesPYC gene encoding the amino acid sequence of SEQ ID NO: 3, an R. etliPYC gene encoding the amino acid sequence of SEQ ID NO: 4, a P.fluorescens PYC gene encoding the amino acid sequence of SEQ ID NOs: 5or 6, a C. glutamicum PYC gene encoding the amino acid sequence of SEQID NO: 7, or an S. meliloti PYC gene encoding the amino acid sequence ofSEQ ID NO: 8; one or more non-native PPC genes such as an E. coli PPCgene encoding the amino acid sequence of SEQ ID NO: 10, an M.thermoautotrophicum PPC gene encoding the amino acid sequence of SEQ IDNO: 11, or a C. perfringens PPC gene encoding the amino acid sequence ofSEQ ID NO: 12; one or more non-native AAT genes such as an E. coli aspCgene encoding the amino acid sequence of SEQ ID NO: 16 or an S.cerevisiae AAT2 gene encoding the amino acid sequence of SEQ ID NO: 15;one or more non-native ADC genes such as an S. avermitilis panD geneencoding the amino acid sequence of SEQ ID NO: 17 (and/or comprising thecoding region of the nucleotide sequence set forth in any one of SEQ IDNOs: 130, 145, 146, or 147), a C. acetobutylicum panD gene encoding theamino acid sequence of SEQ ID NO: 18 (and/or comprising the codingregion of the nucleotide sequence set forth in SEQ ID NO: 131), an H.pylori ADC gene encoding the amino acid sequence of SEQ ID NO: 133(and/or comprising the coding region of the nucleotide sequence setforth in SEQ ID NO: 132), a Bacillus sp. TS25 ADC gene encoding theamino acid sequence of SEQ ID NO: 135 (and/or comprising the codingregion of the nucleotide sequence set forth in SEQ ID NO: 134), a C.glutamicum ADC gene encoding the amino acid sequence of SEQ ID NO: 137(and/or comprising the coding region of the nucleotide sequence setforth in SEQ ID NO: 136), or a B. licheniformis ADC gene encoding theamino acid sequence of SEQ ID NO: 139 (and/or comprising the codingregion of the nucleotide sequence set forth in any one of SEQ ID NOs:138, 148, 149,150, or 151); one or more non-native BAAT or gabT genessuch as an S. kluyveri pyd4 gene encoding the amino acid sequence of SEQID NO: 21 (and/or comprising the coding region of the nucleotidesequence set forth in SEQ ID NO: 142), an S. avermitilis BAAT geneencoding the amino acid sequence of SEQ ID NO: 22 (and/or comprising thecoding region of the nucleotide sequence set forth in SEQ ID NO: 140),an S. avermitilis gabT gene encoding the amino acid sequence set forthin SEQ ID NO: 23, or an S. cerevisiae UGA1 gene encoding the amino acidsequence set forth in SEQ ID NO: 24 (and/or comprising the coding regionof the nucleotide sequence set forth in SEQ ID NO: 141); one or morenon-native 3-HPDH genes such as an E. coli ydfG gene encoding the aminoacid sequence of SEQ ID NO: 27 (and/or comprising the coding region ofthe nudeotide sequence set forth in SEQ ID NO: 143) or an S. cerevisiaeYMR226C gene encoding the amino acid sequence of SEQ ID NO: 129 (and/orcomprising the coding region of the nucleotide sequence set forth in SEQID NO: 144); one or more non-native malonate semialdehyde reductasegenes such as an M. sedula malonate semialdehyde reductase gene encodingthe amino acid sequence set forth in SEQ ID NO: 29 (and/or comprisingthe coding region of the nucleotide sequence set forth in SEQ ID NO:343); one or more non-native HIBADH genes such as an A. faecalis M3Agene encoding the amino acid sequence set forth in SEQ ID NO: 28, a P.putida KT2440 or E23440 mmsB gene encoding the amino acid sequence setforth in SEQ ID NO: 30 or SEQ ID NO: 31, respectively, or a P.aeruginosa PAO1 mmsB gene encoding the amino acid sequence set forth inSEQ ID NO: 32; and/or one or more non-native 4-hydroxybutyratedehydrogenase genes such as an R. eutropha H16 4hbd gene encoding theamino acid sequence set forth in SEQ ID NO: 33 or a C. kluyveri DSM 555hbd gene encoding the amino acid sequence set forth in SEQ ID NO: 34.

Example 3A-0: Enzymatic Activity Assays for Modified Yeast StrainsExpressing Aspartate/Malonate Semialdehyde Pathway Genes Preparation ofCrude Cell-Free Extracts (CFE) for Enzyme Assays:

The indicated cells herein from shake flask or bioreactor cultures werecollected by centrifugation, the supernatant discarded, and the cellpellet stored at −80° C. as described above. For preparation of CFE, thecells pellets were thawed, washed with phosphate-buffered saline (PBS)and again collected by centrifugation. The supernatant was discarded andthe cell pellet was resuspended in an approximately equal volume oflysis buffer containing 1% Protease Inhibitor Cocktail, P8215 fromSigma) in 2.0 mL microcentrifuge tubes. Approximately 300 μL of 0.5 mmzirconia beads (BioSpec) were added, and cell lysis was performed onFastPrep)-24 disruptor (MP Biomedicals) for 3 rounds at setting 6/20seconds. Sample tubes were cooled on ice for 5 minutes between eachround. After lysis, the samples were centrifuged at maximum speed in amicrocentrifuge for 15 minutes at 4° C. The supernatants weretransferred to fresh 1.5 mL tubes and kept on ice. Total proteinconcentrations in the lysates were determined using the Bio-Rad proteinassay reagent (Bradford assay) and bovine serum albumin as the standard,according to the instructions provided by the manufacturer.

Pyruvate Carboxylase (PYC) Activity:

Pyruvate carboxylase activity in CFE of the indicated cells herein wasdetermined as follows. A stock reaction mix solution was prepared that,when combined with CFE in the assay reaction mixture, provides thefollowing final concentration of components: Tris (pH 8.0), 100 mM;NaHCO₃, 10 mM; MgCl₂, 5 mM; NADH, 0.2 mM; ATP, 1 mM; acetyl CoA, 1 mM;pyruvate, 1 mM; biotin (if required by the PYC enzyme being assayed), 5μM; bovine heart malate dehydrogenase, 0.02 units/mL. 270 μL of thismixture was added to the wells of a 96-well microtiter plate and 30 μLof an appropriately diluted CFE was added to start the reaction.Consumption of NADH was monitored at 340 nm using a SpectraMax 340 PCplate reader. Pyruvate carboxylase activity is expressed as nmoles NADHconsumed/sec/mg protein.

Phosphoenolpyruvate Carboxylase (PPC) Activity:

Phosphoenolpyruvate Carboxylase (PPC) activity in CFE may be determinedas follows. A stock reaction mix solution is prepared that, whencombined with CFE in the assay reaction mixture, provides the followingfinal concentration of components: Tris (pH 8.0), 100 mM; NaHCO₃, 10 mM;MgCl₂, 5 mM; NADH, 0.1 mM; acetyl CoA, 0.5 mM; phosphoenolpyruvate, 3.3mM; bovine heart (or porcine heart) malate dehydrogenase, 0.02 units/mL.270 μL of this mixture is added to the wells of a 96-well microtiterplate and 30 μL of an appropriately diluted CFE is added to start thereaction. Consumption of NADH is monitored at 340 nm using a SpectraMax340 PC plate reader.

Aspartate Aminotransferase (AAT) Activity:

Aspartate aminotransferase activity in CFE of the indicated cells hereinwas determined as follows. A stock reaction mix solution was preparedthat, when combined with CFE in the assay reaction mixture, provides thefollowing final concentration of components: 100 mM Tris (pH 8.0), 100mM; NaHCO₃, 10 mM; MgCl₂, 5 mM; NADH, 0.1 mM; aspartate, 1 mM;α-ketoglutarate, 1 mM; and malate dehydrogenase, 0.02 units/mL. In someassays, the stock reaction mixture also contained pyridoxal 5′-phosphate(0.1 mM). 270 μL of this mixture was added to the wells of a 96-wellmicrotiter plate and 30 μL of an appropriately diluted CFE was added tostart the reaction. Consumption of NADH was monitored at 340 nm using aSpectraMax 340 PC plate reader. Aspartate aminotransferase activity isexpressed as nmoles NADH consumed/sec/mg protein.

Aspartate Decarboxylase (ADC) Activity:

Aspartate Decarboxylase activity in CFE of the indicated cells hereinwas determined as follows. 165 μL of 100 mM NH₄Ac buffer (pH 6.8), and25 μL of 80 mM aspartate were added to each well of a 96-well mictotiterplate thermostatted at 37° C. The reaction was initiated by adding 10 μLof CFE. At different time intervals (5, 10, 15, 20, 25, 30, 40, 60minutes), 20 μL of sample was withdrawn from the reaction mixture andadded to 180 μL of quenching buffer (2% formic acid plus 2 mg/L of ¹³Clabeled β-Alanine as internal standard). After filtration, β-Alanine inthe sample was analyzed by LC/MS/MS. Slopes were obtained from β-Alaninevs time plots. Activity was calculated by dividing the slope by totalcellular protein concentration in the reaction. ADC activity isexpressed as μmoles β-alanine formed/sec/g protein.

A modified ADC assay was used in some experiments. In these cases, ADCactivity in CFE of the indicated cells herein was determined as follows.110 μL of 100 mM NH₄Ac buffer (pH 7.6), and 80 μL of 25 mM aspartate(after neutralizing with NaOH) were added to each well of a 96-wellmictotiter plate thermostatted at 40° C. The reaction was initiated byadding 10 μL of CFE. At different time intervals (2, 4, 6, 8, 10minutes), 20 μL of sample was withdrawn from the reaction mixture andadded to 180 μL of quenching buffer (2% formic acid with 2 mg/L of ¹³Clabeled β-Alanine as internal standard or quenched in 2% formic acid andthen transferred 1:10 into 20% methanol/80% water with 2 mg/L of ¹³Clabeled β-Alanine as internal standard). After filtration, β-Alanine inthe sample was analyzed by LC/MS/MS. Slopes were obtained from β-Alaninevs time plots. Activity was calculated by dividing the slope by totalcellular protein concentration in the reaction. ADC activity isexpressed as μmoles β-alanine formed/sec/g protein.

β-Alanine Aminotransferase (BAAT) Activity:

β-Alanine aminotransferase (BAAT) activity in CFE was determined asfollows. 190 μL of a reaction mixture containing 100 mM of NH₄HCO₃ (pH7.6), 8 mM α-ketoglutarate, 0.5 mM acetyl-CoA, 0.1 mMpyridoxal-5′-phosphate, and 200 mM β-alanine was added to a 96 wellmicrotiter plate at room temperature. The reaction was initiated byadding 10 μL of CFE. Samples of 20 μL each were taken at 2, 4, 6, 8, 10,12, 15, and 20 minutes and added to 75 μL of quenching buffer (2.5%formic acid). Samples were neutralized and pH controlled by adding 5 μl10 M NaOH and 50 μl 100 mM NaCO₃ (pH 10). Filtered samples werederivatized by mixing, at injection, with OPA (o-phthaldialdehyde)reagent, 10 mg/mL (Agilent Technologies 5061-3335). Glutamatederivatized with OPA was quantified after HPLC separation byfluorescence detection (excitation at 340 nm; emission at 460 nm).Samples of 15 μL were injected onto an analytical reverse phase GeminiC18 column with 5 μm packing (Phenomonex 150×4.6 mm). The column wasequilibrated in 62.5% 20 mM phosphate buffer (pH 7.8) (A) and 37.5%methanol (B). Linear gradients were as follows: ramp to 40% B, 0-0.3min; 40% B, 0.3-1 min; ramp to 85% B, 1-1.75 min; 85% B, 1.75-2.25 min;ramp to 37.5%, 2.25-3 min; 37.5% B, 3-4 min. The flow rate was 2 mL/min.Standard curves of glutamate in reaction buffer were used to determinethe concentration of the samples. Slopes were obtained from [glutamate]vs time plots. Activity was calculated by dividing the slope with totalcellular protein concentration in the reaction.

3-HP Dehydrogenase (3-HPDH) Activity:

3-HP dehydrogenase activity in CFE of the indicated cells herein wasdetermined as follows. 190 μL of diluted (typically a 100× dilution) CFEin 100 mM of NH₄HCO₃ (pH 7.6) and NADPH were added to each well of a96-well mictotiter plate thermostatted at 37° C. The reaction wasinitiated by adding 10 μL of 60 mM malonate semialdehyde (MSA, freshlyprepared in 10 mM H₂SO₄ from 200 mM MSA stock solution in 10 mM H₂SO₄).Samples of 20 μL each were taken at 1, 2, 4, 6, 8, 10, and 12 minutes,and quenched in 80 μL of boiling water. After cooling, mix 75 μL ofquenched mixture with 75 μL of buffer containing 2 mM NH₄Ac (pH 6.8) and3 mg/L of ¹³C labeled 3-HP. After filtration, 3-HP in the sample wasquantified by LC/MS/MS. Slopes were obtained from 3-HP vs time plots.Activity was calculated by dividing the slope by total cellular proteinconcentration in the reaction. 3-HP dehydrogenase activity is expressedas nmoles NADPH formed/sec/mg protein.

A modified 3-HPDH assay was used in some experiments. In these cases,3-HPDH activity in CFE of the indicated cells herein was determined asfollows. Malonate semi-aldehyde reduction was measured by following thedisappearance of the NADPH over time at 340 nm. Malonate semi-aldehydewas synthesized in-house according to the protocol developed by Yamadaand Jacoby (Yamada, E. W., Jacoby, W. B., 1960, Direct conversion ofmalonic semialdehyde to acetyl-coenzyme A, Journal of BiologicalChemistry, Volume 235, Number 3, pp. 589-594). The assay was conductedin a 96 well micro-plate, and the final volume was 200 μL. The reactionwas started by adding 30 μL of CCE into 170 μL of assay buffer (2 mMmalonate semi-aldehyde, 100 mM Tris pH 8.0 and 0.5 mM NADPH). Absorbanceat 340 nm was followed on a micro-plate reader (Spectra Max 340PC,Molecular Devices LLC, Sunnyvale, Calif.) for 10 minutes at roomtemperature (˜25° C.). One unit of 3-HPDH activity is defined as theamount of enzyme necessary to oxidize 1 pmol of NADPH in one minute inthe presence of malonate semi-aldehyde.

Example 3A-1: Insertion Vectors for Expressing Aspartate Decarboxylase(ADC) at the Adh1202 Locus

Several aspartate decarboxylase genes were codon-optimized forexpression in I. orientalis and synthesized by GeneArt® (Burlingame,Calif., USA) resulting in the plasmids listed in the Table 7. Thesynthetic genes arrived in the vector pMA-T and can be elicited from thevector via XbaI and PacI restriction digest. The restriction fragmentcan then be cloned into the same sites in pMIBa107 placing the geneunder the control of the PDC promoter and terminator, and allowingintegration to occur at the I. orientalis adh1202 locus.

TABLE 7 Transformant constructs Construction Gene SEQ IntegrationPlasmid Gene Source Gene Number ID NO construct Transformant 1051387Helicobacter pylori P56065 132 pWTY10- yWTY1-1 0033-1 yWTY1-2 1051391Bacillus sp. TS25 ZY440006.gene3 134 pWTY10- yWTY1-5 0033-2 yWTY1-61051389 Corynebacterium Q9X4N0 136 pWTY10- yWTY1-9 glutamicum 0033-3yWTY1-10 1051388 Clostridium P58285 131 pWTY10- yWTY1-13 acetobutylicum0033-4 yWTY1-14 1051390 Bacillus licheniformis Q65I58 138 pWTY10-yWTY1-17 0033-5 yWTY1-18 1045172 Streptomyces 130 pWTY10- yWTY1-25avermitilis 0033-7 yWTY1-26

Plasmids 1045172, 105387, 105388, 105389, 105390, and 105391 weredigested with XbaI and PacI and run on a 1.3% agarose gel using TBEbuffer. Fragments of 400-500 bp from each digest corresponding to theADC (panD) gene were excised from the gel and extracted from the agaroseusing a QIAQUICK® Gel Extraction Kit (Qiagen).

Plasmid pMIBa107 was digested with XbaI and PacI, treated with calfintestinal phosphatase (New England Biolabs) and the vector bandpurified after agarose gel electrophoresis in TBE buffer. The XbaI andPacI digested panD fragments were ligated into this purified pMIBa107vector using T4 DNA ligase and a Quick ligation kit (New EnglandBiolabs) according to the manufacturer's instructions. The ligationproducts were transformed into XL10-GOLD ULTRA cells (AgilentTechnologies) according to manufacturer's instructions. Transformantswere plated onto 2× YT+amp plates and incubated at 37° C. overnight.Twenty-four transformants from each reaction were picked to 2× YT+ampplates. Mini-prep DNA from four each of the resulting transformants wasscreened by ApaI, NcoI and SacI digestion. Clones yielding the desiredband sizes were confirmed to be correct by DNA sequencing and weredesignated as shown in Table 7. The resulting plasmids allow integrationof each ADC gene at the adh1202 locus with the expression cassetteoriented in the forward direction.

Approximately 10 μg each of each integration construct was digested withApaI and KpnI and run on a 1% agarose gel using 89 mM Tris base-89 mMBoric Acid-2 mM disodium EDTA (TBE) buffer. Fragments of approximately4450 bp for each plasmid were excised from the gel and extracted usingthe QIAquick gel extraction kit (Qiagen) according to the manufacturer'sinstructions. The concentration of the purified products was found to bebetween 39-138 ng/ul. 0.39-1.4 μg of the fragments from the integrationconstructs (digested with ApaI and Kpn I) were transformed into I.orientalis CNB1 as described above. Transformants were plated onto uraselection media and incubated at 37° C., re-streaked onto ura selectionmedia, and incubated at 37° C. overnight. Genomic DNA was prepared fromthe URA3+ colonies and checked by PCR to confirm integration. Primers0611718 and 0611632 were used to amplify a 2.5 kbp fragment to confirmintegration. Each PCR reaction contained 2.5 μL of genomic DNA, 12.5 μMeach of primers 0611718 and 0611632, 1× Crimson Taq™ Reaction Buffer(New England Biolabs), 0.2 mM dNTP mix, 0.625 Units Crimson Taq™ DNApolymerase (New England Biolabs) in a final volume of 25 μL. Theamplification reactions were incubated in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for 1 cycle at 95° C. for 30 seconds;30 cycles each at 95° C. for 30 seconds, 50° C. for 30 seconds, and 68°C. for 3 minutes; and 1 cycle at 68° C. for 10 minutes.

Two URA3+ confirmed transformants for each construct were designated asshown in Table 7. These strains are heterozygous at the adh1202 for theindicated ADC gene with expression driven by the PDC promoter andterminator from I. orientalis.

PanD expression and enzyme activity from strains listed in Table 7 (andstrain MBin500, supra, as negative control) was tested. Overnightcultures of each strain were grown overnight in YPD ON at 37° C.,diluted 1:50 into 25 mL of fresh YPD in 125 mL baffled flask at 37° C.,and grown to an OD₆₀₀ ˜2-8. The cell pellets were then used to prepareCFE, which was then assayed for ADC activity as described supra.Representative results are shown in Table 8.

TABLE 8 Transformant enzyme activity data Gene SEQ Strain Source of ADCGene ID NO ADC activity MBin500 N/A N/A 0 (control) yWTY1-1 Helicobacterpylori 132 0 yWTY1-2 yWTY2-5 Bacillus sp. TS25 134 0.31 yWTY2-6 0.15yWTY3-9 Corynebacterium glutamicum 136 0.31 yWTY3-10 0.19 yWTY4-13Clostridium acetobutylicum 131 0.37 yWTY4-14 0.23 yWTY5-17 Bacilluslicheniformis 138 0.56 yWTY5-18 0.61 yWTY7-25 Streptomyces avermitilis130 0.23 yWTY7-26 0.30

Next, homozygous versions of yWTY5-17 and yWTY7-25 were created. First,ura-derivatives yWTY5-17 and yWTY7-25 were isolated as described above.Genomic DNA was prepared from the FOA-resistant colonies and checked byPCR as describe above to confirm loss of the URA3 selectable marker.Primers 0611718 and 0611632 were used to amplify a 2.4 kbp fragment forintegration with the ura marker present and 1100 bp fragment in theabsence of the ura marker. Ura-strains of yWTY5-17 and yWTY7-25 thatyielded a PCR fragment of 1100 bp with primers 0611718 and 0611632 weredesignated MIBa331 and MIBa332, respectively.

10 μg each of pWTY10-0033-5 and pWTY10-0033-7 were digested with ApaI,KpnI, and NcoI and run on a 1% agarose gel using 89 mM Tris base-89 mMBoric Acid-2 mM disodium EDTA (TBE) buffer. Fragments of approximately4450 bp for each plasmid were excised from the gel and extracted using aQIAQUICK® Gel Extraction Kit (Qiagen). The purified fragments frompWTY10-0033-5 and pWTY10-0033-7 were transformed into MIBa331 andMIBa332, respectively as described above. Transformants were plated ontoura selection media and incubated overnight at 37° C., and thenre-streaked onto ura selection media and incubated at 37° C. overnight.Genomic DNA was prepared and Crimson Taq™ PCRs were run to confirmintegration as described above. Primers 0611718 and 0611632 amplify a2.4 kbp fragment for integration with the ura marker present, andamplify a 1100 bp fragment in the absence of the ura marker.Transformants of MIBa331 and MIBa332 that yielded PCR fragments of 1100bp and 2.4 kbp with primers 0611718 and 0611632 were saved anddesignated MIBa338 and MIBa337, respectively. MIBa337 is homozygous forADC gene from S. avermitilis at the adh1202 loci and MIBa338 ishomozygous for the ADC gene from B. licheniformis at the adh1202 loci.Both strains have the control of the respective ADC gene under the PDCpromoter and terminator from I. orientalis.

ADC expression and enzyme activity from the panD homozygous strainsMIBa337 and MIBa338 were compared to the heterozygous panD strainsyWTY5-17 and yWTY7-25. Cultures were grown in YPD overnight at 37° C.,and then diluted 1:50 into 25 mL of fresh YPD in 125 mL baffled flasksat 37° C. and grown to an OD₆₀₀ ˜2-8. The cell pellets were used toprepare CFE, which was then assayed for ADC activity as described above.Representative results for two independent experiments are shown inTable 9A.

TABLE 9A Transformant enzyme activity data ADC Gene activity SEQ (Exp.1, Strain Source of panD Gene Allele Type ID NO Exp. 2) MBin500 N/A N/AN/A 0, 0 (control) yWTY5-17 Bacillus licheniformis heterozygous 138 0.6, 0.29 MIBa338 Bacillus licheniformis homozygous 138   0, 0.22yWTY7-25 Streptomyces heterozygous 130 0.19, 0.13 avermitilis MIBa337Streptomyces homozygous 130 0.28, 0.19 avermitilis

The results of a third independent experiment to compare ADC activity inCFE prepared from strains MIBa337 and MIBa338 are shown in Table 9B.

TABLE 9B Transformant enzyme activity data Gene SEQ ADC Strain Source ofpanD gene Allele type ID NO activity MBin500 N/A N/A N/A 0 (control)yWTY5-17 Bacillus licheniformis heterozygous 138 0.218 MIBa338 Bacilluslicheniformis homozygous 138 0.453 yWTY7-25 Streptomyces heterozygous130 0.087 avermitilis MIBa337 Streptomyces homozygous 130 0.188avermitilis

SDS-PAGE analysis of the samples above indicated that panD expressionfrom MIBa338 was the highest among these strains.

Strains MBin500, MIBa337 and MIBa338 were evaluated in bioreactors for3-HP production, using the method described herein. Control strainMBin500 produced no detectable 3-HP (average of two independentfermentations). Strain MIBa337 produced 1.33 g/L 3-HP (one fermentationperformed) and strain MIBa338 produced 3.15 g/L 3-HP (average of threeindependent fermentations). Individual fermentations of strains MIBa337and MIBa338 were further compared with respect to their 3-HP productionperformance and ADC activity (Table 10). In order to account fordifferences in cell mass in these fermentations, the 3-HP productionperformance is reported in Table 10 as 3-HP concentration per unit ofcell mass (expressed as [g/L 3-HP]/[g/L dry cell weight]). The resultsshow the improved ADC activity and 3-HP production performance whenusing the Bacillus licheniformis panD gene (strain MIBa338) vs. theStreptomyces avermitilis panD gene (strain MIBa337).

TABLE 10 3-HP production performance and ADC activity in strains MIBa337and MIBa338 MIBa337 MIBa338 Fermentation ADC Activity 3HP/ Activity 3HP/time (hr) (mmol/min/g prot) DCW (mmol/min/g prot) DCW 11 0.005 0.000.021 0.024 22 0.011 0.05 0.055 0.131 31 0.003 0.04 0.029 0.159 48 0.0010.05 0.018 0.142

Example 3A-2: Insertion Vectors for Expressing β-AlanineAminotransferase (BAAT) or 3-Hydroxypropionic Acid Dehydrogenase(3-HPDH) at the Adh1202 Locus

I. orientalis codon-optimized versions of BAAT from S. avermitilis, UGA1from S. cerevisiae, PYD4 from S. kluyveri, YMR226c from S. cerevisiae,and ydfG from E. coli were synthesized by GeneArt® resulting in theplasmids listed below. The synthetic genes arrived in the vector pMA-Tand can be elicited from the vector via digest using XbaI and Pac I. Thedigested fragment can then be cloned into the same sites in pMIBa107,placing the gene under the control of the PDC promoter and terminatorand allowing integration to occur at the adh1202 locus.

TABLE 11 Transformant constructs Con- struction SEQ Integration Trans-Plasmid Gene Gene Source ID NO construct formant 1045169 gabT S.cerevisiae 141 pMIBa122 MIBa310 (UGA1) 1045170 BAAT S. avermitilis 140pMIBa121 MIBa309 1045171 BAAT S. kluyveri 142 pMIBa124 MIBa312 (PYD4)1045173 3-HPDH S. cerevisiae 144 pMIBa123 MIBa311 (YMR226c) 10451683-HPDH E. coli 143 pMIBa120 MIBa308 (ydfG)

Plasmids 1054168, 1054169, 1054170, 1054171, 1054172, and 1054173 weredigested with XbaI and PacI and run on a 1% agarose gel using 89 mM Trisbase-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer. Fragments of 761(ydfG) bp from 1045168, 1430 (UGA1) bp from 1045169, 1370 (BAAT) bp from1045170, 1442 (PYD4) bp from 1045171, or 814 (YMR226c) bp from 1045173were excised from the gel and extracted from the agarose using aQIAQUICK® Gel Extraction Kit (Qiagen). Plasmid pMIBa107 was digestedwith XbaI and PacI followed by treatment with CIP resulting in a 7.9 kbplinear fragment. The digest was purified using a QIAQUICK® PCRPurification Kit (Qiagen). The digested fragments of ydfG, UGA1, BAAT,PYD4, or YMR226c were then ligated into pMIBa107 (digested with XbaI andPacI and treated with CIP) using T4 DNA ligase as described herein. Theligation products were transformed into One Shot® TOP10 ChemicallyCompetent E. coli cells (Invitrogen) according to manufacturer'sinstructions. Transformants were plated onto 2× YT+amp plates andincubated at 37° C. overnight. Several of the resulting transformantswere screened by digestion with XbaI and Pac I. Clones yielding thedesired band sizes were confirmed to be correct by DNA sequencing anddesignated pMIBa120, pMIBa121, pMIBa122, pMIBa123, and pMIBa124 forydfG, BAAT, UGA1, YMR226c, or PYD4, respectively. The resulting plasmidsallow integration of the desired gene at the adh1202 locus with theexpression cassette oriented in the forward direction.

The integration constructs in Table 11 were used to integrate the genesof interest codon-optimized for expression in I. orientalis into theadh1202 locus under the control of the PDC promoter and terminator. Theexpression cassette also contains a URA3 selectable marker to allowselection of transformants within a ura-host as described herein. Theexpression cassettes and adh1202 homology regions are flanked by ApaIand KpnI restriction sites to allow release of the fragment from theplasmid.

15 μg each of integration constructs in Table 11 were digested withApaI, KpnI, and NcoI and run on a 1% agarose gel using 89 mM Trisbase-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer. Digestion withNcoI breaks up the vector backbone and makes it easier to extract thefragment of interest from the agarose gel. Fragments of 4884 bp, 5493bp, 5553 bp, 4937 bp, and 5565 bp from pMIBa120, pMIBa121, pMIBa122,pMIBa123, and pMIBa124, respectively, were excised from the gel andextracted from the agarose using a QIAQUICK® Gel Extraction Kit(Qiagen). The concentration of the purified products was found to bebetween 80-120 ng/μL. 0.8-1.2 μg of the restriction fragments frompMIBa120-4 were transformed into I. orientalis CNB1 (ura-) as describedherein. The transformants then were plated onto ura selection media andgrown at room temperature for 60 hours. Transformants were re-streakedonto ura selection media and incubated at 37° C. overnight.

Several transformants of each were checked by colony PCR to confirmintegration. Correct integration was confirmed by using primer pairsthat check the 5′ and 3′ ends of the integrations and are listed below.The primer 0611717 anneals in the PDC promoter in the reverse direction,while primer 0611225 anneals in the URA3 selectable marker in theforward direction. Primers 0611631 and 0611632 anneal outside of thesite of integration going in the forward and reverse directions,respectively; primers 0611717 and 0611631 amplify a 976 bp fragment incorrect integrants; primers 0611225 and 0611632 amplify a 1.4 kbpfragment in correct integrants; and primers 0611631 and 0611632 amplifya 2.7 kbp fragment indicating a wildtype chromosome and will amplifyfragments ˜5 kbp for integrations. To create genomic DNA, one colony ofeach transformant was incubated in 50 μL of 0.05 U/μL lyticase (Sigma,St. Louis, Mo., USA) in TE at 37° C. for 30 minutes followed incubationat 95° C. for 10 minutes. PCRs were run as described herein to confirmintegration. One transformant of each heterozygous integrant thatyielded PCR fragments of 976 bp with 0611717 and 0611631, 1.4 kbp with0611225 and 0611632, and 2.7 kbp with 0611631 and 0611632 was saved anddesignated MIBa308, MIBa309, MIBa310, MIBa311, and MIBa312 as shown inTable 11.

Cultures of the transformants MIBa308, MIBa309, MIBa310, MIBa311, andMIBa312 were grown overnight in YPD at 37° C. Cultures were then diluted1:50 into 25 mL of fresh YPD in 125 mL baffled flask at 37° C. and grownto an OD₆₀₀ ˜4-10. Samples of the cells were analyzed for proteinexpression by SDS-PAGE using the methods described herein. CFE were alsoprepared from cell pellets from the cultures, and 3HP dehydrogenaseactivity was measured in the CFE using the method described herein.Expression of UGA1 and PYD4 from strains MIBa310 and MIBa312,respectively, was detected by SDS-PAGE by the appearance of a ˜53 KDaband that was absent in strains not integrated for either gene.Expression of BAAT in MIBa309 was not detected by SDS-PAGE under theseconditions. Table 12A shows the 3-HP dehydrogenase (3-HPDH) activity inthe CFE of the strains.

TABLE 12A Transformant enzyme activity data Gene Gene SEQ 3-HPDH StrainOverexpressed Gene Source ID NO activity MBin500 N/A N/A N/A 0.28, 0.24MIBa310 gabT S. cerevisiae 141 0.39 (UGA1) MIBa309 BAAT S. avermitilis140 0.39 MIBa312 BAAT S. kluyveri 142 0.45 (PYD4) MIBa311 3-HPDH S.cerevisiae 144 1.1 (YMR226c) MIBa308 3-HPDH E. coli 143 0.67 (ydfG)

In an independent experiment using improved assay conditions, the BAATactivity in CFE prepared from strains MBin500 (control), MIBa310,MIBa309 and MIBa312 was compared. The results of this experiment areshown in Table 12B.

TABLE 12B Transformant enzyme activity data. Gene Gene SEQ StrainOverexpressed Gene Source ID NO BAAT activity MBin500 N/A N/A N/A 0.67MIBa310 gabT S. cerevisiae 141 9.05 (UGA1) MIBa309 BAAT S. avermitilis140 0.42 MIBa312 BAAT S. kluyveri 142 105.85 (PYD4)

The plasmids pMIBa120-4 (supra) contain NotI restriction sites thatflank the expression cassette as follows: PDC promoter, gene of interest(BAAT or 3-HPDH), PDC terminator, and the URA3 selection marker. Thehomology for integration at the adh1202 locus is outside of the NotIrestriction sites. These plasmids all have the expression cassette inforward orientation.

New plasmids were constructed with the expression cassette oriented inthe reverse direction to allow ease of screening homozygous integrationstrains. Plasmids pMIBa120-4 were digested with NotI and run on a 1%agarose gel using 89 mM Tris base-89 mM Boric Acid-2 mM disodium EDTA(TBE) buffer. Fragments of 3.5 kbp (pMIBA120), 4.2 kbp (pMIBa121), 4.2kbp (pMIBA122), 3.5 kbp (pMIBa123), and 4.2 kbp (pMIBA124) were excisedfrom the gel and extracted from the agarose using a QIAQUICK® GelExtraction Kit (Qiagen). Each of these fragments was ligated into the5.2 kbp linear NotI/CIP treated pHJJ76-no ura using T4 DNA ligase asdescribed herein. The ligation products were transformed into One Shot®TOP10 Chemically Competent E. coli cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened by XbaI and KpnI digestion. Clones yieldingthe desired band sizes were designated pMIBa131, pMIBa132, pMIBa133,pMIBa134, and pMIBa135 for UGA1, YMR226c, PYD4, ydfG, and BAAT,respectively. The resulting plasmids allow integration of the desiredgene at the adh1202 locus with the expression cassette oriented in thereverse direction.

Ura-derivatives of MIBa308-12 were isolated as described herein. SeveralFOA-resistant colonies for MIBA308-12 were colony purified twice bygrowing on YPD plates 37° C. Genomic DNA was prepared from theFOA-resistant colonies and checked by PCR to confirm loss of URA3selectable marker as described herein. Primers 0611631 and 0611632anneal outside of the site of integration going in the forward andreverse directions, respectively. Primer 0611718 anneals in the PDCterminator upstream of the ura selectable marker; primers 0611632 and0611631 amplify a 2.7 kbp fragment for a wildtype chromosome; andprimers 0611718 and 0611632 amplify a 2.4 kbp fragment for anintegration with the ura marker present and 1100 bp fragment in theabsence of the ura marker. One ura-strain of MIBa308-12 that yielded thePCR fragments of 1100 bp with 0611718 and 0611632, and 2.7 kbp with0611631 and 0611632 was saved and designated MIBa314 (ura-strain ofMIBa310), MIBa315 (ura-strain of MIBa312), MIBa316 (ura-strain ofMIBa311), MIBa326 (ura-strain of MIBa308), and MIBA328 (ura-strain ofMIBa309).

10-15 μg each of pMIBa131, pMIBa132, pMIBa133, and pMIBa135 weredigested with ApaI, KpnI, and NcoI and run on a 1% agarose gel using 89mM Tris base-89 mM Boric Acid-2 mM disodium EDTA (TBE) buffer. Digestionwith NcoI facilitates extraction of the fragment of interest from theagarose gel. Fragments of 5553 bp, 4937 bp, 5565 bp, 5493 bp frompMIBa131-3, pMIBa135, respectively, were excised from the gel andextracted from the agarose using a QIAQUICK® Gel Extraction Kit(Qiagen). The concentration of the purified products was found to bebetween 67-80 ng/μL. 0.67-0.8 μg of the restricted fragments frompMIBa131-3, and pMIBa135 were transformed into MIBa314, MIBa316,MIBa315, or MIBa328 as described herein. Transformants were plated ontoura selection media and incubated overnight at 37° C., and thenre-streaked onto ura selection media and incubated overnight at 37° C.overnight. Genomic DNA was prepared from the URA3+ colonies and checkedby PCR as described herein to confirm integration of the secondexpression cassette, making the strain homozygous for the gene ofinterest Primers 0611718 and 0611632 amplify a 1100 bp fragment for thefirst integration as described above, and primers 0611632 and 0611717amplify a 814 bp fragment for the second integration in the reverseorientation. URA3+ transformants of each lineage that amplified a 1100bp fragment with 0611718 and 0611632 and a 814 bp fragment with 0611717and 0611632 were designated MIBA317, MIBA318, MIBA319, and MIBa329 (seeTable 13).

TABLE 13 Transformant genotypes Strain Genotype MIBa317 adh1202Δ::(PDC_(promo)-Opt.ScYMR226c, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScYMR226c, URA3) ura3-/ura3- MIBa318adh1202Δ::(PDC_(promo)-Opt.ScUGA1, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScUGA1, URA3) ura3-/ura3- MIBa319adh1202Δ::(PDC_(promo)-Opt.SkPYD4, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.SkPYD4, URA3) ura3-/ura3- MIBa329 adh1202Δ::(PDC_(promo)-Opt.SaBAAT, URA3-Scar/ adh1202Δ:: (PDC_(promo)-Opt.SaBAAT,URA3) ura3-/ura3-

The expression and enzyme activities from strains homozygous orheterozygous for YMR226c, UGA1, PYD4, and BAAT were determined.Overnight cultures of MIBA309-12, MIBa317-9 and MIBa329 were grown inYPD ON at 37° C., and then diluted 1:50 into 25 mL of fresh YPD in 125mL baffled flask at 37° C. and grown to an OD₆₀₀ ˜4-10. Samples of thecells were analyzed for protein expression by SDS-PAGE using the methoddescribed herein. CFE were also prepared from cell pellets from thecultures, and 3HP dehydrogenase activity was measured in the CFE usingthe method described herein. Based on SDS-PAGE results, strains MIBa310,MIBa318, MIBa312 and MIBa319 contained a protein with a mass of ˜53 KDa(the expected size of the proteins encoded by UGA1 or PYD4 genes). Theband corresponding to this protein was not observed in the SDS-PAGEanalysis of strain MBin500. In addition, expression of UGA1 and PYD4from homozygous strains MIBa318 and MIBa319, respectively, was greaterthan the corresponding heterozygous strains MIBa310 or MIBa312 (asjudged by the SDS-PAGE analysis). BAAT expression was not detected (bySDS-PAGE) in strains MIBa309 or MIBa329 under these conditions. Table14A shows the 3-HP dehydrogenase (“3-HPDH”) activity in CFE of strainsMBin500, MIBa311 and MIBa317.

TABLE 14A Transformant enzyme activity data Gene Gene SEQ Over- ID3-HPDH Strain expressed NO Source Allele Type activity MBin500 N/A N/AN/A N/A 0.13 (control) MIBa311 3-HPDH 144 S. cerevisiae heterozygous1.49 (YMR226c) MIBa317 3-HPDH 144 S. cerevisiae homozygous 2.85(YMR226c)

In an independent experiment using improved assay conditions, the BAATactivity in CFE prepared from strains MBin500 (control), MIBa319 andMIBa329 was compared. The results of this experiment are shown in Table14B.

TABLE 14B Transformant enzyme activity data Gene Gene SEQ ID StrainOverexpressed NO Source BAAT activity MBin500 N/A N/A N/A 0.67 (control)MIBa319 BAAT 142 S. kluyveri 228.01 (PYD4) MIBa329 BAAT 140 S.avermitilis 0.38

Ura-derivatives of strains MIBa317, MIBa318, and MIBa319 were isolatedas described herein. Several FOA-resistant colonies for MIBa317, MIBa18,and MIBa19 were colony purified by growing on YPD plates at 37° C.Genomic DNA was prepared from the FOA-resistant colonies and checked byPCR as described herein to confirm loss of URA3 selectable marker.Primers 0611718 and 0611632 amplify a 1100 bp fragment indicating thefirst integration as described above, and primers 0611632 and 0611717amplify a 814 bp fragment indicating the presence of the secondintegration in the reverse orientation. Primers 0611718 and 0611631amplify a 2.6 kbp fragment indicating the second integration with theura marker and a 1200 bp fragment without the ura marker. Ura-strains ofMIBa317 and MIBa318 that yielded PCR fragments of 1100 bp with 0611718and 0611632, 814 bp with 0611632 and 0611717, or 1200 bp with 0611718and 0611631 were saved and designated MIBa320 and MIBa321, respectively.When the ura marker was removed from MIBa319 a possible gene conversionevent occurred resulting in MIBa322 as indicated by PCR (no 2.7 kbpfragment with 0611632 and 0611631 primers or 814 bp fragment with0611632 and 0611717, but amplified 1100 bp fragment with 0611718 and0611632) so that both expression cassettes were oriented in the forwarddirection.

Example 3A-3: Construction of Left-Hand Fragments of Insertion Vectorsfor Expressing Aspartate 1-Decarboxylase (ADC), β-AlanineAminotransferase (BAAT), and 3-Hydroxypropionic Acid Dehydrogenase(3-HPDH) at the Pdc Locus

Left-Hand Fragment Containing S. avermitilis ADC (SEQ ID NO: 130) and S.avermitilis BAAT (SEQ ID NO: 140)

To allow insertion of a gene for expression between the ENO1 promoterand PDC terminator regions, the pMhCt068 vector was digested with XbaIand PacI, treated with 10 units calf intestinal phosphatase (New EnglandBiolabs) at 37° C. for 30 minutes, and purified by 0.9% agarose gelelectrophoresis in TAE buffer, and an approximately 6.1 kbp band wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

The Saccharomyces kluvyeri BAAT (pyd4) gene (SEQ ID NO: 142) was thenamplified with primers 0611196 and 0611186 which contain restrictionsites for subsequent subcloning. The PCR reaction (50 μL) contained 1 μLof a 1 to 50 dilution of a mini-prep of plasmid containing the S.kluvyeri pyd4 gene, 1× Pfx Amplification Buffer (Invitrogen), 100 pmoleach of primers 0611196 and 0611186, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1.5 μL DMSO and 2.5 units of PlatinumR Pfx DNA Polymerase(Invitrogen). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 95° C. for 2 minutesfollowed by 34 cycles each at 95° C. for 30 seconds, 40.8° C. for 30seconds, and 72° C. for 1 minute 30 seconds, with a final extension at72° C. for 5 minutes. Following thermocycling, the PCR reaction productswere separated by 0.9% agarose gel electrophoresis in TAE buffer wherean approximately 1428 bp PCR product was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

The pyd4 PCR product generated above was digested with XbaI and PacI andpurified by 0.9% agarose gel electrophoresis in TAE buffer, and anapproximately 1.4 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. This purified DNA was cloned into the XbaIand PacI restricted pMhCt068 vector described above in a ligationreaction (20 μL) containing 1× Quick ligation buffer (New EnglandBiolabs), 100 ng XbaI/PacI pMhCt068 vector, 70.5 ng Xba I/PacI pyd4insert, and 1 μL Quick T4 DNA ligase (New England Biolabs). The ligationreaction was incubated for 5 min at room temperature, and then cooled onice. 5 μL of this reaction was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired PCRproducts by digestion with XbaI and PacI as described herein, with oneverified isolate designated “left+pyd4#1”.

A polynucleotide encoding the S. avermitilis ADC of SEQ ID NO: 17 andcodon-optimized for expression in E. coli was amplified with the primers0611376 and 0611377 (note that primer 0611376 results in the removal ofthe “T” base on the 5′ end of the NheI restriction site followinginsertion via In-Fusion into pMhCt068, which removes an unwanted ATGstart codon present in the initial pMhCt068 clone). The PCR reaction (50μL) contained 1 μL of a 1 to 50 dilution of a mini-prep of plasmidcontaining the S. avermitilis ADC gene optimized for E. coli, 1×ThermoPol Reaction buffer (New England Biolabs), 100 pmol each ofprimers 0611376 and 0611377, 200 μM each of dATP, dCTP, dGTP, and dTTP,2 μL 100 mM MgSO₄, and 2 units of Vent_(R)® (exo-) DNA polymerase (NewEngland Biolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 94° C. for 2 minutesfollowed by 34 cycles each at 94° C. for 30 seconds, 54° C. for 30seconds, and 72° C. for 1 minute, with a final extension at 72° C. for10 minutes. Following thermocycling, the PCR reaction products wereseparated by 0.9% agarose gel electrophoresis in TAE buffer where anapproximately 420 bp PCR product was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The “left+pyd4#1” plasmid then was digested with NheI and AscI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and purified by 0.9% agarose gel electrophoresis inTAE buffer, and an approximately 7.5 kbp band was excised from the geland purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions. The purified PCR fragmentabove containing the S. avermitilis ADC gene optimized for E. coli wasdigested with NheI and AscI and purified by 0.9% agarose gelelectrophoresis in TAE buffer, and an approximately 420 bp band wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions. Theresulting fragment then was ligated into the linearized “left+pyd4#1”vector in a ligation reaction (20 μL) containing 1× Quick ligationbuffer (New England Biolabs), 100 ng NheI/AscI “left+pyd4#1” vector, 31ng of the NheI and AscI digested panD insert, and 1 μL Quick T4 DNAligase (New England Biolabs). The ligation reaction was incubated for 5min at room temperature and then placed on ice. 5 μL of this reactionwas used to transform SoloPack Gold SuperCompetent Cells (AgilentTechnologies) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion of the S. avermitilis ADC gene optimized for E. coli bydigestion with AscI and Pvu II as described herein. A clone yielding thedesired band sizes was confirmed to be correct by DNA sequencing anddesignated pMhCt070.

The plasmid pMhCt070 served as the base vector for the addition of ADCand BAAT homologs that had been codon-optimized for expression in theyeast host. pMhCt070 was digested with XbaI and PacI, treated with 10units calf intestinal phosphatase (New England Biolabs) at 37° C. for 30minutes, and purified by 0.9% agarose gel electrophoresis in TAE buffer,and an approximately 6.5 kbp band was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. An XbaI and PacI digested fragmentdescribed above containing a polynucleotide that encodes the S.avermitilis BAAT (SEQ ID NO: 140) and codon-optimized for expression inI. orientalis was ligated into the pMhCt070 cut vector as follows: Aligation reaction (20 μL) containing 1× Quick ligation buffer (NewEngland Biolabs), 42 ng of the XbaI and PacI digested pMhCt070 vector, 4μL of the codon-optimized S. avermitilis BAAT XbaI and PacI digestedinsert, and 1 μL Quick T4 DNA ligase (New England Biolabs). The ligationreaction was incubated for 5 min at room temperature, and then placed onice. 5 μL of this reaction was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired BAAT ORFby XbaI, PacI, and Eco RV digestion as described herein. A cloneyielding the desired band sizes was confirmed to be correct by DNAsequencing and designated pMhCt072.

The S. avermitilis ADC gene codon-optimized for expression in E. coli inpMhCt072 then was replaced with a version codon-optimized for expressionin I. orientalis (SEQ ID NO: 130). The I. orientalis codon-optimized ADCgene (SEQ ID NO: 130) and desired additional restriction sites andflanking DNA were amplified with primers 0611378 and 0611379. The PCRreaction (50 μL) contained 1 μL of a 1 to 50 dilution of mini-prep ofthe plasmid containing the codon-optimized S. avermitilis panD(GeneArt®), 1× ThermoPol Reaction buffer (New England Biolabs), 100 pmoleach of primers 0611378 and 0611379, 200 μM each of dATP, dCTP, dGTP,and dTTP, 2 μL 100 mM MgSO₄, and 2 units of Vent_(R)® (exo-) DNApolymerase (New England Biolabs). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific) programmed for one cycle at 94° C.for 2 minutes followed by 34 cycles each at 94° C. for 30 seconds, 54°C. for 30 seconds, and 72° C. for 45 seconds, with a final extension at72° C. for 10 minutes. Following thermocycling, the PCR reactionproducts were separated by 1% agarose gel electrophoresis in TAE bufferwhere an approximately 420 base pair PCR product was excised from thegel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions.

5 μL of a mini-prep of pMhCt072 was digested with XbaI and PacI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and purified by 1% agarose gel electrophoresis in TAEbuffer, and an approximately 7.5 kbp band was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions. The isolated PCR product containingthe codon-optimized S. avermitilis ADC gene from above was added to thevector in the following IN-FUSION™ Advantage PCR Cloning Kit (Clontech)reaction: the 10 μL reaction volume was composed of 6 μL of the pMhCt072digested and purified vector, 1 μL of the purified codon-optimized panDPCR product, 1× In-Fusion reaction buffer (Clontech) and 1 μL ofIN-FUSION™ enzyme (Clontech). The reaction was incubated at 37° C. for15 minutes, 50° C. for 15 minutes, and then placed on ice. The reactionwas diluted with 40 μL of TE buffer and 2.5 μL was used to transformSoloPack Gold SuperCompetent Cells (Agilent Technologies) according tothe manufacturer's instructions. Transformants were plated onto 2×YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion of thedesired PCR products by NheI, AscI, and ClaI digestion. A clone yieldingthe desired band sizes was confirmed to be correct by DNA sequencing anddesignated pMhCt074 (FIG. 7).

pMhCt074 is a left-hand PDC targeting construct containing the PDCpromoter driving expression of the codon-optimized S. avermitilis ADC(panD, SEQ ID NO: 130), the TAL terminator, the ENO1 promoter drivingexpression of the codon-optimized S. avermitilis BAAT (SEQ ID NO: 140),the RKI terminator, the I. orientalis URA3 promoter and the 5′ fragmentof the I. orientalis URA3 ORF.

Left-Hand Fragment Containing S. avermitilis ADC (SEQ ID NO: 130) and S.Kluyveri BAAT (SEQ ID NO: 142)

To create a left-hand DNA construct that expresses the S. kluyveri BAAT(pyd4), a fragment from XbaI and PacI digestion containing the S.kluyveri BAAT sequence codon-optimized for expression in I. orientalis(SEQ ID NO: 142, supra) was ligated into the pMhCt070 digested vectorabove as follows: A ligation reaction (20 μL) containing 1× Quickligation buffer (New England Biolabs), 42 ng of the XbaI and PacIdigested pMhCt070 vector, 4 μL of the codon-optimized S. kluyveri pyd4insert digested with XbaI and PacI, and 1 μL Quick T4 DNA ligase (NewEngland Biolabs). The ligation reaction was incubated for 5 minutes atroom temperature, and then placed on ice. 5 μL of this reaction was usedto transform SoloPack Gold SuperCompetent Cells (Agilent Technologies)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion of thedesired pyd4 ORF by XbaI, PacI, and Eco RV digestion. A clone yieldingthe desired band sizes was confirmed to be correct by DNA sequencing anddesignated pMhCt073.

The plasmid pMhCt073 contains the desired S. kluyveri BAAT (pyd4)sequence codon-optimized for expression in I. orientalis but does notcontain the desired S. avermitilis ADC (panD) sequence codon-optimizedfor expression in I. orientalis. To move in this ORF, 5 μL of amini-prep of pMhCt073 was digested with XbaI and PacI, treated with 10units calf intestinal phosphatase (New England Biolabs) at 37° C. for 30minutes, and purified by 1% agarose gel electrophoresis in TAE buffer.An approximately 7.5 kbp band was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. The isolated PCR product containing thecodon-optimized S. avermitilis panD (supra) was added to the vector inthe following IN-FUSION™ Advantage PCR Cloning Kit (Clontech) reaction:the 10 μL reaction volume was composed of 6 μL of the pMhCt073 digestedand purified vector, 1 μL of the purified codon-optimized panD PCRproduct, 1× In-Fusion reaction buffer (Clontech) and 1 μL of IN-FUSION™enzyme (Clontech). The reaction was incubated at 37° C. for 15 minutes,50° C. for 15 minutes, and then placed on ice. The reaction was dilutedwith 40 μL of TE buffer and 2.5 μL was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired PCRproducts by NheI, AscI, and ClaI digestion. A clone yielding the desiredband sizes was confirmed to be correct by DNA sequencing and designatedpMhCt076.

Plasmid pMhCt076 is a left-hand PDC targeting construct containing thePDC promoter driving expression of the S. avermitilis ADCcodon-optimized for expression in I. orientalis (panD, SEQ ID NO: 130),the TAL terminator, the ENO1 promoter driving expression of the S.kluyveri BAAT codon-optimized for expression in I. orientalis (pyd4, SEQID NO: 142), the RKI terminator, the I. orientalis URA3 promoter and the5′ fragment of the I. orientalis URA3 ORF.

Sequencing determined that plasmid pMhCt076 contains an A to Tnucleotide change at about 200 bp into the PDC promoter, and a G to Tnucleotide change ˜⅔ of the way thru the PDC promoter that are presentin the pMhCt068 parent vector (supra).To address any concern aboutpotential alteration in gene expression, a construct similar to pMhCt076but containing the corrected PDC promoter was cloned as described below.

5 μL of a mini-prep of pMhCt082 was digested with Nhe and PacI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and purified by 0.9% agarose gel electrophoresis inTAE buffer, and an approximately 4.7 kbp band was excised from the geland purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions. 4 μL of a mini-prep ofpMhCt076 was digested with NheI and PacI and purified by 0.9% agarosegel electrophoresis in TAE buffer, and an approximately 3.3 kbp band wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions. Thepurified 4.7 kbp vector and 3.3 kbp insert were then ligated together ina ligation reaction (20 μL) containing 1× Quick ligation buffer (NewEngland Biolabs), 3 μL pMhCt082 vector, 6 μL pMhCt076 insert, and 1 μLQuick T4 DNA ligase (New England Biolabs). The ligation reaction wasincubated for 5 min at room temperature, and then placed on ice. 5 μL ofthis reaction was used to transform ONE SHOT® TOP10 chemically competentE. coli cells (Invitrogen) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at roomtemperature for three days. Several of the resulting transformants werescreened for proper insertion of the desired PCR products by digestionwith StuI and Not I. A clone yielding the desired band sizes wasconfirmed to be correct by DNA sequencing and designated pMhCtO83 (FIG.8).

Plasmid pMhCt083 is identical to pMhCt076 except at the former containsthe correct PDC promoter sequence, while the latter has one A to Tnucleotide change and one G to T nucleotide change described above.Testing showed no difference in panD enzymatic activity from strainsexpressing S. avermitilis panD from integration of pMhCt076 and pMhCtO77as compared to pMhCt083 and pMhCtO77.

Left-Hand Fragment Containing S. avermitilis ADC (SEQ ID NO: 130) andSaccharomyces cerevisiae gabT (SEQ ID NO: 141)

4 μL of a mini-prep of pMhCt083 was digested with XbaI and PacI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and purified by 0.9% agarose gel electrophoresis inTAE buffer, and an approximately 6.5 kbp band was excised from the geland purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions. A fragment digested withXbaI and PacI containing the Saccharomyces cerevisiae gabTcodon-optimized for expression in I. orientalis (UGA1, SEQ ID NO: 141)was ligated into the pMhCt083 cut vector as follows: A ligation reaction(20 μL) containing 1× Quick ligation buffer (New England Biolabs), 1 μLof the purified pMhCt083 vector digested with XbaI and PacI, 3 μLcodon-optimized S. cerevisiae UGA1 XbaI and PacI digested insert, and 1μL Quick T4 DNA ligase (New England Biolabs). The ligation reaction wasincubated for 5 min at room temperature and then the tube was placed onice. 5 μL of this reaction was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired BAAT ORFby XbaI and Bg/II digestion. A clone yielding the desired band sizes wasdesignated pMhCt087 (FIG. 9).

Plasmid pMhCt087 is a left-hand PDC targeting construct containing thePDC promoter driving expression of the S. avermitilis ADCcodon-optimized for expression in I. orientalis (panD, SEQ ID NO: 130),the TAL terminator, the ENO1 promoter driving expression of the S.cerevisiae gabT codon-optimized for expression in I. orientalis (UGA1,SEQ ID NO: 141), the RKI terminator, the I. orientalis URA3 promoter andthe 5′ fragment of the I. orientalis URA3 ORF.

Example 3A-4: Construction of Right-Hand Fragments of Insertion Vectorsfor Expressing Aspartate 1-Decarboxylase (ADC), β-AlanineAminotransferase (BAAT), and 3-Hydroxypropionic Acid Dehydrogenase(3-HPDH) at the Pdc Locus

Right-Hand Fragment Containing E. coli 3-HPDH (SEQ ID NO: 143)

2 μg of a mini-prep of pMhCt069 (supra) was digested with XbaI and PacI,treated with 10 units calf intestinal phosphatase (New England Biolabs)at 37° C. for 30 minutes, and purified by 0.9% agarose gelelectrophoresis in TAE buffer, and an approximately 2.2 kbp band wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions. Afragment digested with XbaI and PacI containing E. coli 3-HPDH genecodon-optimized for expression in I. orientalis (ydfG, SEQ ID NO: 143)was ligated into the pMhCt069 cut vector as follows: A ligation reaction(20 μL) containing 1× Quick ligation buffer (New England Biolabs), 2 μLof the purified pMhCt069 vector digested with XbaI and PacI, 4 μL of thecodon-optimized E. coli ydfG insert digested with XbaI and PacI, and 1μL Quick T4 DNA ligase (New England Biolabs). The ligation reaction wasincubated for 5 min at room temperature, and then placed on ice. 5 μL ofthis reaction was used to transform SoloPack Gold SuperCompetent Cells(Agilent Technologies) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at roomtemperature for three days. Several of the resulting transformants werescreened for proper insertion of the desired ydfG ORF by XbaI, PacI, andEco RV digestion. A clone yielding the desired band sizes was confirmedto be correct by DNA sequencing as described herein and designatedpMhCt075 (FIG. 10).

Plasmid pMhCt075 contains the 3′ fragment of the I. orientalis URA3 ORF,the URA3 terminator from I. orientalis followed by the URA3 promoter(for later looping out of the URA3 marker), E. coli 3-HPDH genecodon-optimized for expression in I. orientalis (ydfG, SEQ ID NO: 143)driven by the I. orientalis TDH3 promoter, and the I. orientalis PDCterminator regions.

Right-Hand Fragment Containing Saccharomyces cerevisiae 3-HPDH (SEQ IDNO: 144)

A fragment digested with XbaI and PacI containing the S. cerevisiaeYMR226C gene codon-optimized for expression in I. orientalis (supra) wasligated into the pMhCtO69 cut vector as follows: A ligation reaction (20μL) containing 1× Quick ligation buffer (New England Biolabs), 2 μL ofthe purified pMhCt069 vector digested with XbaI and PacI, 4 μL of thecodon-optimized S. cerevisiae 3-HPDH (YMR226C, SEQ ID NO: 144) insertdigested with XbaI and PacI, and 1 μL Quick T4 DNA ligase (New EnglandBiolabs). The ligation reaction was incubated for 5 min at roomtemperature, and then placed on ice. 5 μL of this reaction was used totransform SoloPack Gold SuperCompetent Cells (Agilent Technologies)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at room temperature for three days.Several of the resulting transformants were screened for properinsertion of the desired YMR226C ORF by XbaI, PacI, and Eco RVdigestion. A clone yielding the desired band sizes was confirmed to becorrect by DNA sequencing and was designated pMhCt077 (FIG. 11).

Plasmid pMhCt077 contains the 3′ fragment of the I. orientalis URA3 ORF,the URA3 terminator from I. orientalis followed by the URA3 promoter(for later looping out of the URA3 marker), the S. cerevisiae 3-HPDHgene codon-optimized for expression in I. orientalis (YMR226C, SEQ IDNO: 144) driven by the I. orientalis TDH3 promoter, and the I.orientalis PDC terminator regions.

Example 3A-5: Heterozygous and Homozygous Yeast Strains ExpressingAspartate 1-Decarboxylase (ADC), β-Alanine Aminotransferase (BAAT), and3-Hydroxypropionic Acid Dehydrogenase (3-HPDH) at the PDC Locus

Examples 3A-3 and 3A-4 above describe the construction of variousleft-hand or right-hand constructs for targeting expression of threeectopic genes simultaneously to the I. orientalis PDC locus. Prior totransformation, approximately 10 μg of each construct (one desiredleft-hand construct and one desired right-hand construct) was digestedwith NotI to release the desired transforming DNA from the pUC18backbone vector; for most digestions, the restriction enzyme PvuI wasalso included with the NotI digestion. Restriction enzyme PvuI digeststhe pUC18 vector fragment approximately in half, making separation ofthe larger, desired DNA fragment more facile by gel electrophoresis. Thelarger, expression cassette containing band was separated from the pUC18backbone DNA by gel electrophoresis, excised from the gel, and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. 30 μL elution buffer was used for theelution step. An equimolar ratio of one left-hand and one right-handconstruct totaling 10 μL were used to transform the I. orientalis strainCNB1 or appropriate derivative. Transformants were selected on uraselection plates and placed at 37° C. for growth. The next day,approximately twelve transformants were picked and restreaked for singlecolonies to ura selection plates and grown at 37° C. The following day,a single colony was picked from each of the streaks generated by eachinitial transformant and restreaked to ura selection plates for singlecolonies. After another night of growth at 37° C., a final single colonywas picked from each streak and restreaked to a ura selection plate andgrown overnight at 37° C. After this second round of single colonypurification and outgrowth, genomic DNA was prepared for use in PCR toverify the desired targeted integration occurred as described above. Fortargeting to PDC using the left- and right-hand constructs, verificationof the desired integration event was determined using primers 0611814,0611554, and 0611555. Primer 0611554 binds in the I. orientalis genomicDNA just 3′ of the PDC terminator region present in the right-hand PDCtargeting constructs; primer 0611555 binds in PDC ORF and amplifiestoward stop; and primer 0611814 binds near the 3′ end of the TDH3promoter region present in the right-hand constructs and amplifies inthe 3′ direction. Generation of an approximately 1.9 kbp band from PCRsthat contained primers 0611814 and 0611554 indicated the occurrence ofthe desired integration event at the PDC locus. Generation of anapproximately 1.4 kbp band from PCRs that contained primers 0611555 and0611554 indicated the presence of a wild-type PDC locus. Since thisintegration event is the first targeting event in the diploid I.orientalis CNB1, the desired integrants will show both a 1.9 kbp bandfor primers 0611814 and 0611554 and a 1.4 kbp band from primers 0611555and 0611554. Two independent transformants giving the desired bandpattern for each plasmid were designated as shown in Table 15.

TABLE 15 Transformant genotypes Plasmid Plasmid w/ left- w/ right- handhand Strain fragment fragment Genotype yMhCt002 pMhCt074 pMhCt075pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)- 74/75 #1 Opt.SaBAAT, URA3,TDH3_(promo)-Opt.EcYdfG)/PDC ura3-/ura3- yMhCt004 pMhCt083 pMhCt077pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)- 83/77 #2 Opt.SkPyd4, URA3,TDH3_(promo-) Opt.ScYMR226C)/PDC ura3-/ura3- yMhCt005 pMhCt087 pMhCt077pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)- 87/77 #2 Opt.ScUGA1, URA3,TDH3_(promo)- Opt.ScYMR226C)/PDC ura3-/ura3-

Next, a ura-derivative of yMhCt004 or yMhCt005 was isolated as describedabove. Genomic DNAs from several FOA resistant colonies of each parentstrain were screened by PCR for the desired loop-out event with primers0611815 and 0611817. Primer 0611815 anneals in the RKI terminator of theleft-hand construct and amplifies toward the URA3 promoter. Primer0611817 anneals in TDH3 promoter and amplifies back toward the URA3cassette. The presence of an 828 bp band indicates the presence of onlythe ura3 scar site (a single URA3 promoter left behind after homologousrecombination between the two URA3 promoters in the parent strain) asdesired, while a band of approximately 2.2 kbp indicates the presence ofthe intact URA3 promoter-URA3 ORF-URA3 terminator-URA3 promotercassette, indicating the desired recombination event did not occur. PCRreactions with Crimson Taq™ DNA polymerase (New England Biolabs) werecarried out as described above. One FOA resistant colony from parentstrain yMhCt004, designated yMhCt012, and one FOA resistant colony fromparent strain yMhCt005, designated yMhCt007, gave the desired 828 bpband.

Strains yMhCt012 and yMhCt007 were next transformed to create homozygousstrains with the PDC gene deleted and replaced with expression cassettesfor panD, pyd4, and YMR226C or panD, UGA1, and YMR226C, respectively.Strain yMhCt012 was transformed with linear DNA from pMhCt083 andpMhCt077, while yMhCt007 was transformed with linear DNA from pMhCt087and pMhCt077. After two rounds of single colony purification, genomicDNAs from several transformants from each parent strain were screened byPCR with primers 0611815 and 0611817 as described above. Twoindependently isolated integrants from each parent strain that had boththe 828 bp band (from amplification of the ura3 scar region from theoriginally targeted PDC locus) and the 2.2 kbp band (from integration ofthe URA3 promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette fromthe second integration event on the other chromosome) were designated asshown in Table 16.

TABLE 16 Transformant geneotypes Strain designation Parent strainGenotype yMhCt013 yMhCt012 pdcΔ::(PDC_(promo)-Opt.SaPanD,ENO1_(promo)-Opt.SkPyd4, yMhCt014 URA3-Scar,TDH3_(promo)-Opt.ScYMR226C)/ pdcΔ::(PDC_(promo)-Opt.SaPanD,ENO1_(promo)Opt.SkPyd4- URA3, TDH3_(promo)-Opt.ScYMR226C) ura3-/ura3-yMhCt008 yMhCt007 pdcΔ::(PDC_(promo)-Opt.SaPanD,ENO1_(promo)-Opt.ScUGA1, yMhCt009 URA3-Scar,TDH3_(promo)-Opt.ScYMR226C)/ pdcΔ::(PDC_(promo)-Opt.SaPanD,ENO1_(promo)-Opt.ScUGA1, URA3, TDH3_(promo)-Opt.ScYMR226C) ura3-/ura3-

A ura-derivative of yMhCt008 was isolated as described above. GenomicDNAs from several FOA resistant colonies from the yMHCt008 parent strainwere screened by PCR for the desired loop-out event with the primers0611815 and 0611817 as described herein. The presence of an 828 bp bandindicated the presence of only the ura3 scar site (a single URA3promoter left behind after homologous recombination between the two URA3promoters in the parent strain) as desired. Isolates that showed onlythe 828 bp band were further screened using primers 0611555 and 0611554as described herein. Generation of an approximately 1.4 kbp band fromPCRs that contained primers 0611555 and 0611554 indicated the presenceof a wild-type PDC locus. An isolate lacking this band, indicating thatthe PDC locus on both chromosomes had been lost, was designatedyMhCt010.

Strains were grown in shake flasks and CFE were prepared and assayed foraspartate decarboxylase (ADC) activity and 3-HP dehydrogenase (3-HPDH)activity as described in herein. The experimental results for severalassay sets (denoted as Trials 1-4) are shown in Table 17. The strainsfrom Trial 1 of Table 17 were also analyzed by SDS-PAGE as describedherein. Strain 74/75 #1 and strain yMhCt002 of Trial 1 gave a band inSDS-PAGE analysis at 27 kD that was not present in the control strain ofTrial 1 (MBin500). The size of this protein band is consistent with itsidentity as the protein encoded by the ydfG gene.

TABLE 17 Transformant enzyme activity data Gene ADC 3-HPDH Trial StrainOverexpressed Allele Type activity activity 1 MBin500 N/A N/A Not tested0.25 2 (control) 0.00 0.12 3 0.00 0.23 4 0.00 0.45 4 0.00 0.54 1 74/75#1 ADC (SEQ ID NO: 130) heterozygous Not tested 0.68 3-HPDH (SEQ ID NO:143) BAAT (SEQ ID NO: 140) 1 yMhCt002 ADC (SEQ ID NO: 130) heterozygousNot tested 0.93 3-HPDH (SEQ ID NO: 143) BAAT (SEQ ID NO: 140) 2 yMhCt004ADC (SEQ ID NO: 130) heterozygous 0.26 1.81 4 3-HPDH (SEQ ID NO: 144)0.30 2.20 4 BAAT (SEQ ID NO: 142) 0.24 1.81 2 83/77 #2 ADC (SEQ ID NO:130) heterozygous 0.25 1.80 3-HPDH (SEQ ID NO: 144) BAAT (SEQ ID NO:142) 2 yMhCt005 ADC (SEQ ID NO: 130) heterozygous 0.17 1.51 4 3-HPDH(SEQ ID NO: 144) 0.17 1.26 4 gabT (SEQ ID NO: 141) 0.14 1.24 2 87/77 #2ADC (SEQ ID NO: 130) heterozygous 0.20 1.68 3-HPDH (SEQ ID NO: 144) gabT(SEQ ID NO: 141) 3 yMhCt005 ADC (SEQ ID NO: 130) heterozygous 0.16 1.273-HPDH (SEQ ID NO: 144) gabT (SEQ ID NO: 141) 3 yMhCt008 ADC (SEQ ID NO:130) homozygous 0.80 2.29 4 3-HPDH (SEQ ID NO: 144) 0.25 0.85 4 gabT(SEQ ID NO: 141) 0.42 1.37 3 yMhCt009 ADC (SEQ ID NO: 130) homozygous .045 1.33 4 3-HPDH (SEQ ID NO: 144) 0.22 0.55 4 gabT (SEQ ID NO: 141)0.43 1.15 4 yMhCt013 ADC (SEQ ID NO: 130) homozygous 0.47 0.65 4 3-HPDH(SEQ ID NO: 144) 0.50 0.82 BAAT (SEQ ID NO: 142) 4 yMhCt014 ADC (SEQ IDNO: 130) homozygous 0.34 0.51 4 3-HPDH (SEQ ID NO: 144) 0.54 1.06 BAAT(SEQ ID NO: 142)

The experimental results for another assay set are shown in Table 17(Trial 2). The strains from Trial 2 of Table 17 were also analyzed bySDS-PAGE as described herein. All strains of Trial 2 except MBin500 gavea band at 29 kD in the SDS-PAGE analysis. The size of this protein bandis consistent with its identity as the protein encoded by the YMR226cgene. Strains yMhCt005 and 87/77 #2 for Trial 2 gave a band at 53 kDthat was not present in the three other samples for this trial. The sizeof this protein band is consistent with its identity as the proteinencoded by the UGA1 gene.

The experimental results for another assay set are shown in Table 17(Trial 3). The strains from Trial 3 of Table 17 were also analyzed bySDS-PAGE as described herein. All strains of Trial 3 except MBin500 gavea band at 53 kD and a band at 29 kD in the SDS-PAGE analysis. The sizesof these protein bands are consistent with the proteins encoded by theUGA1 and YMR226c genes, respectively. Strains MBin500 and yMhCt005 ofTrial 3 showed a band at 64 kD in the SDS-PAGE analysis that was absentin yMhCt008 and yMhCt009 for this trial. The size of this protein bandis consistent with its identity as the protein encoded by the nativepyruvate decarboxylase (PDC) gene in I. orientalis CNB1.

The experimental results for another assay set are shown in Table 17(Trial 4). The strains from Trial 4 of Table 17 were also analyzed bySDS-PAGE as described herein. All strains of Trial 4 except MBin500 gavea band at 29 kD The size of this protein band is consistent with itsidentity as the protein encoded by the YMR226c gene. Strains yMhCt005,yMhCt008, and yMhCt009 of Trial 4 showed a band at 53 kD. The size ofthis band is consistent with the protein encoded by the UGA1 gene.Strains yMhCt013, and yMhCt014 of Trial 4 showed a faint band at 53 kDThe size of this band is consistent with the protein encoded by the PYD4gene. Strains MBin500, yMhCt004, and yMhCt005 of Trial 4 showed a bandat 64 kD that was absent in strains yMhCt008, yMhCt009, yMhCt013, andyMhCt014. The size of this protein band is consistent with its identityas the protein encoded by the native pyruvate decarboxylase (PDC) genein I. orientalis CNB1.

Strains MBin500 and yMhCt008 were tested evaluated in bioreactors for3-HP production, using the method described herein. Control strainMBin500 produced no detectable 3-HP (average of two independentfermentations). Strain yMhCt008 produced 2.45 g/L 3-HP (average oftwelve independent fermentations).

Example 3A-6: Yeast Strains Expressing β-Alanine Aminotransferase (BAAT)or 3-Hydroxypropionic Acid Dehydrogenase (3-HPDH) at the Adh1202 Locus,and Expressing Aspartate 1-Decarboxylase (ADC), β-AlanineAminotransferase (BAAT), and 3-Hydroxypropionic Acid Dehydrogenase(3-HPDH) at the Pdc Locus

20 μg of pMhCtO77, pMhCt083, and pMhCt087 (supra) were digested withNotI and PvuI and then run on a 1% agarose gel using 89 mM Tris base-89mM Boric Acid-2 mM disodium EDTA (TBE) buffer. NotI digested fragmentsof 3815 bp, 5332 bp, or 5320 bp from pMhCt077, pMhCt083, and pMhCt087,respectively, were excised from the gel and extracted from the agaroseusing a QIAQUICK® Gel Extraction Kit (Qiagen). 560 ng of NotI digestedpMhCtO77 and 560 ng of NotI digested pMhCt083 or pMhCt087 weretransformed into strains MIBa320, MIBa321, and MIBa322. MIBa320 wastransformed with pMhCt077/83 and pMhCt077/87 combinations. MIBa321 wastransformed with pMhCtO77/87 and MIBa322 was transformed withpMhCt077/83 as described herein. Transformants were plated onto uraselection media and incubated for approximately 60 hours at roomtemperature. Transformants were re-streaked onto ura selection media andincubated at 37° C. overnight. Genomic DNA was prepared from the URA3+colonies and checked by PCR as described herein to confirm integrationof the expression cassette. The primer pair 611814 and 611554 amplify a1.9 kbp fragment indicating integration. The primer pair 611555 and611554 amplify a 1.4 kbp fragment indicating a wildtype locus. One URA3+transformant of each lineage that amplified PCR fragments of 1.9 kbpwith 611554 and 611814 and 1.4 kbp with 611555 and 611554 was saved;these were designated MIBa323, MIBa324, MIBa325, and MIBa327 (see Table18 for genotypes). Promoters and terminators were derived from I.orientalis genes.

TABLE 18 Transformant genotypes Strain designation Genotype MIBa323adh1202Δ::(PDC_(promo)-Opt.ScYMR226c, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScYMR226c, URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD; ENO1_(promo)-Opt.ScUGA1, URA3,TDH3_(promo)-Opt.ScYMR226c)/PDC ura3-/ura3- MIBa324adh1202Δ::(PDC_(promo)-Opt.ScYMR226c, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScYMR226c, URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.SkPyd4, URA3,TDH3_(promo)-Opt.ScYMR226c)/PDC ura3-/ura3- MIBa325adh1202Δ::(PDC_(promo)-Opt.ScUGA1, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScUGA1, URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD; ENO1_(promo)-Opt.ScUGA1, URA3,TDH3_(promo)-Opt.ScYMR226c)/PDC ura3-/ura3- MIBa327adh1202Δ::(PDC_(promo)-Opt.SkPyd4, URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.SkPyd4, URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.SkPyd4, URA3,TDH3_(promo)-Opt.ScYMR226c)/PDC ura3-/ura3-

Strains were grown in shake flasks and CFE were prepared and assayed for3-HP dehydrogenase (3-HPDH) activity as described herein. The resultsare shown in Table 19.

TABLE 19 Transformant enzyme activity data Gene 3-HPDH StrainOverexpressed activity MBin500 N/A 0.14 (control) MIBa314 gabT (SEQ IDNO: 141) 0.09 MIBa318 gabT (SEQ ID NO: 141) 0.41 MIBa321 gabT (SEQ IDNO: 141) 0.08 MIBa325 gabT (SEQ ID NO: 141) 1.15 ADC (SEQ ID NO: 130)3-HPDH (SEQ ID NO: 144) MIBa315 BAAT (SEQ ID NO: 142) 0.12 MIBa319 BAAT(SEQ ID NO: 142) 0.17 MIBa322 BAAT (SEQ ID NO: 142) 0.09 MIBa327 BAAT(SEQ ID NO: 142) 0.98 ADC (SEQ ID NO: 130) 3-HPDH (SEQ ID NO: 144)MIBa316 3-HPDH (SEQ ID NO: 144) 0.48 MIBa317 3-HPDH (SEQ ID NO: 144)2.15 MIBa320 3-HPDH (SEQ ID NO: 144) 1.07 MIBa323 3-HPDH (SEQ ID NO:144) 2.66 ADC (SEQ ID NO: 130) gabT (SEQ ID NO: 141) MIBa324 3-HPDH (SEQID NO: 144) 2.12 ADC (SEQ ID NO: 130) BAAT (SEQ ID NO: 142)

Ura-derivatives of MIBa323, MIBa324, MIBa325 and MIBa327 were isolatedas described herein. Genomic DNA was prepared from the FOA-resistantcolonies and checked by PCR as described herein to confirm loss of URA3selectable marker. Primer 0611815 anneals in the RKI terminator of theleft-hand construct and amplifies toward the URA3 promoter, and primer0611817 anneals in TDH3 promoter and amplifies back toward the URA3cassette. The presence of an 828 bp band indicates the presence of onlythe URA3 scar site (a single URA3 promoter left behind after homologousrecombination between the two URA3 promoters in the parent strain) asdesired, while a band of approximately 2.2 kbp indicates the presence ofthe intact URA3 promoter-URA3 ORF-URA3 terminator-URA3 promotercassette, indicating the desired recombination event did not occur.Ura-strains of MIBa323, MIBa324, MIBa325, and MIBa327, that yielded PCRfragments of 828 bp with primers 0611815 and 0611817 were saved anddesignated MIBa335, MIBa333, MIBa334, and MIBa336, respectively.

Strains MIBa333 and MIBa334 were transformed with the fragments frompMhCt077 and pMhCt087, and strains MIBa335 and MIBa336 were transformedwith the fragments from pMhCt077 and pMhCt083 as described above in thesection on MIBa320-2 transformations. Transformants were selected for bygrowth on ura selection media as described herein. Genomic DNA wasprepared from the URA3+ colonies and checked by PCR as described hereinto confirm integration of the expression cassette. Primer 0611815anneals in the RKI terminator of the left-hand construct and amplifiestoward the URA3 promoter. Primer 0611817 anneals in TDH3 promoter andamplifies back toward the URA3 cassette. The presence of an 828 bp bandindicates the presence of only the URA3 scar site (a single URA3promoter left behind after homologous recombination between the two URA3promoters in the parent strain) as desired for the first integration,and a band of approximately 2.2 kbp indicates the presence of the intactURA3 promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette for thesecond integration. Primer pair 0611815 and 0611816 amplifies a 625 bpfragment when the ura marker is present. Primers 0611555 and 0611554amplify a 1.4 kbp fragment when the PDC locus is present. Homozygousintegrants should not amplify a fragment with these primers. One URA3+transformant of each lineage that amplified PCR fragments of 828 bp and2.2 kbp with primers 0611815 and 0611817, 625 bp with primers 0611815and 0611816 and no fragment with primers 0611555 and 0611554 was saved;these were designated MIBa340, MIBa341, MIBa345, and MIBa348 (see Table20A). Promoters and terminators were derived from I. orientalis genes.

TABLE 20A Transformant genotypes Strain Designation Genotype MIBa345adh1202Δ::(PDC_(promo)-Opt.ScYMR226c URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScYMR226c URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD ENO1_(promo)-Opt.ScUGA1, URA3-Scar,TDH3_(promo)-Opt.ScYMR226c)/pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.ScUGA1, URA3, TDH3_(promo)-Opt.ScYMR226c) ura3-/ura3- MIBa348adh1202Δ::(PDC_(promo)-Opt.ScYMR226c URA3-Scar)/adh1202Δ::(PDC_(promo)-Opt.ScYMR226c URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.SkPyd4, URA3-Scar,TDH3_(promo)-Opt.ScYMR226c)/pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.SkPyd4, URA3, TDH3_(promo)-Opt.ScYMR226c) ura3-/ura3- MIBa340adh1202Δ::(PDC_(promo)-Opt.ScUGA1 URA3-Scar)/adh1202Δ::(PDC_(promo)Opt.ScUGA1 URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.ScUGA1, URA3-Scar,TDH3_(promo)-Opt.ScYMR226c)/pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.ScUGA1, URA3, TDH3_(promo)-Opt.ScYMR226c) ura3-/ura3- MIBa341adh1202Δ::(PDC_(promo)-Opt.SkPyd4 URA3-Scar)/adh1202Δ::(PDC_(promo)Opt.SkPyd4 URA3-Scar)pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.SkPyd4, URA3-Scar,TDH3_(promo)-Opt.ScYMR226c)/pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.SkPyd4, URA3, TDH3_(promo)-Opt.ScYMR226c) ura3-/ura3-

The aspartate 1-decarboxylase (ADC), beta-alanine aminotransferase(BAAT) and 3-HP dehydrogenase (3-HPDH) activities in CFE prepared fromstrains MBin500 (control), MIBa345, MIBa348, MIBa340 and MIBa341 werecompared. The results of this experiment are shown in Table 20B.

TABLE 20B Transformant enzyme activity data ADC BAAT 3-HPDH Strain GenesOverexpressed Gene Sources Activity Activity Activity MBin500 N/A N/A0.002 0.61 0.4 (control) MIBa345 YMR226c (SEQ ID NO: 144) S. cerevisiae0.194 14.37 79.5 ADC (SEQ ID NO: 130) S. avermitilis UGA1 (SEQ ID NO:141) S. cerevisiae MIBa348 YMR226c (SEQ ID NO: 144) S. cerevisiae 0.169173.67 76.7 ADC (SEQ ID NO: 130) S. avermitilis PYD4 (SEQ ID NO: 142) S.kluyveri MIBa340 YMR226c (SEQ ID NO: 144) S. cerevisiae 0.239 19.51 64.8ADC (SEQ ID NO: 130) S. avermitilis UGA1 (SEQ ID NO: 141) S. cerevisiaeMIBa341 YMR226c (SEQ ID NO 144) S. cerevisiae 0.22 386.92 65.5 ADC (SEQID NO: 130) S. avermitilis PYD4 (SEQ ID NO: 142) S. kluyveri

Example 3A-7: Left-Hand Fragments of Insertion Vectors with MultipleNucleotide Sequences for Expressing Aspartate 1-Decarboxylase (ADC) atthe Adh1202 Locus

Constructs were designed to incorporate four copies of nucleotidesencoding an ADC (SEQ ID NO: 17) at the I. orientalis adh1202 locus. In asimilar approach to that described herein for the PDC locus, a left-handand a right-hand construct were designed to allow homologousrecombination. The general design of the integration vectors and desiredrecombination event is shown in FIG. 3. This approach was also used forexpression of an alternative ADC (SEQ ID NO: 139) as described in theexamples below.

To prevent recombination from occurring between the multiple copies ofthe nucleotide sequences encoding the same ADC sequence, four distinctnucleotide sequences codon-optimized for expression in I. orientalis(SEQ ID NOs: 130, 145, 146, and 147) were designed to encode the sameADC sequence of SEQ ID NO: 17. Additionally, since the initial set ofconstructs was designed to target the ald5680 locus of I. orientalis,the adh1202 targeting sequences were incorporated into these vectors ata late step in the cloning. The ald5680 constructs can be used to targeta second locus in an I. orientalis CNB1 strain already homozygous forectopic four copies of panD at adh1202 with four additional copies ofpanD at ald5680.

The left-hand ald5680 targeting vector was constructed as follows. A PCRproduct containing the sequence just 5′ of the ald5680 ORF, along withthe desired additional restriction sites and flanking DNA for cloningwas amplified with primers 0612271 and 0612272. The PCR reaction (50 μL)contained 1 μL of a 1 to 50 dilution of pHJJ75 mini-prep plasmid DNA(FIG. 23), 1× iProof™ HF buffer (Bio-Rad Laboratories), 100 pmol each ofprimers 0612271 and 0612272, 200 μM each of dATP, dCTP, dGTP, and dTTP,and 1 unit of iProof™ High Fidelity DNA polymerase (Bio-RadLaboratories). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 32 cycles each at 98° C. for 10 seconds, 59° C. for 20seconds, and 72° C. for 45 seconds, with a final extension at 72° C. for10 minutes. Following thermocycling, the PCR reaction products wereseparated by 1% agarose gel electrophoresis in TAE buffer where anapproximately 930 base pair PCR product was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

In order to allow purification of a greater quantity of DNA, the PCRreaction described above was repeated using the purified 930 bp PCRproduct as the template DNA. Five 50 μL reactions were set up andamplified with the conditions described above except that 1 μL of thepurified 930 bp PCR product replaced the pHJJ75 plasmid (supra) astemplate DNA. Following thermocycling, the amplified 930 bp product waspurified as above.

A fragment containing PDC promo-optPanD-ENO1-UGA1 (which contains the I.orientalis codon-optimized S. avermitilis ADC encoding sequence of SEQID NO: 130) was excised from pMhCt087 (supra) via NotI and EcoRIdigestion. 10 μg of a midi-prep of pMhCt087 was digested with NotI andEcoRI and then purified by 0.9% agarose gel electrophoresis in TAEbuffer. An approximately 4.4 kbp band was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

A PCR product containing the 5′ half of the URA3 split marker with thedesired additional restriction sites and flanking DNA for cloning wasamplified using the primers 0612273 and 0612274. The PCR reaction (50μL) contained 1 μL of a 1 to 50 dilution of pMhCt082 mini-prep plasmidDNA (supra), 1× iProof™ HF buffer (Bio-Rad Laboratories), 100 pmol eachof primers 0612273 and 0612274, 200 μM each of dATP, dCTP, dGTP, anddTTP, and 1 unit of iProof™ High Fidelity DNA polymerase (Bio-RadLaboratories). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 32 cycles each at 98° C. for 10 seconds, 59° C. for 20seconds, and 72° C. for 45 seconds, with a final extension at 72° C. for10 minutes. Following thermocycling, the PCR reaction products wereseparated by 1% agarose gel electrophoresis in TAE buffer where anapproximately 960 base pair PCR product was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

To create a recipient vector for the above DNA fragments, the plasmidpUC19 (Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene, 33,103-119) was digested with Hind III and EcoRI, treated with 10 unitscalf intestinal phosphatase (New England Biolabs) at 37° C. for 30minutes, and purified by 0.9% agarose gel electrophoresis in TAE buffer,and an approximately 2.6 kbp band was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The purified 930 bp, 4.4 kbp, and 960 bp DNA fragments from above werethen inserted into the digested pUC19 fragment using an IN-FUSION™Advantage PCR Cloning Kit (Clontech) in a total reaction volume of 10 μLcomposed of 150 ng of the pUC19 vector digested with Hind III and EcoRI,56 ng of the 930 bp DNA containing the ald5680 flanking DNA, 250 ng ofthe PDC promo-optPanD-ENO1-UGA1 fragment from pMhCt087 digested withNotI and EcoRI, 55 ng of the 960 bp PCR product 5′ containing the 5′half of the URA3 split marker, 1× In-Fusion reaction buffer (Clontech)and 1 μL of IN-FUSION™ enzyme (Clontech). The reaction was incubated at37° C. for 15 minutes, 50° C. for 15 minutes, and then placed on ice.The reaction then was diluted with 40 μL of TE buffer and 2.5 μL wasused to transform SoloPack Gold SuperCompetent Cells (AgilentTechnologies) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at roomtemperature for three days. Several of the resulting transformants werescreened for proper insertion of the desired PCR products by SalI andHpaI digestion. A done yielding the desired band sizes was confirmed tobe correct by DNA sequencing and designated pMhCt089.

Next, the UGA1 ORF in pMhCt089 was replaced with the S. avermitilis panDgene codon-optimized for expression in I. orientalis which encodes theADC of SEQ ID NO: 17. S. avermitilis panD version r1 (SEQ ID NO: 145)was synthesized by GeneArt® in the vector pMA-T. The plasmid pMA-T wasdigested with XbaI and PacI and the resulting fragments were separatedby 0.9% agarose gel electrophoresis in TAE buffer, and an approximately434 bp band was excised from the gel and purified using a NUCLEOSPIN)Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions. Plasmid pMhCt089 was digested with XbaI and PacI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and the resulting fragments were separated by 0.9%agarose gel electrophoresis in TAE buffer, and an approximately 4.5 kbpband was excised from the gel and purified using a NUCLEOSPIN® ExtractII Kit (Macherey-Nagel) according to the manufacturer's instructions.The pMhCt089 vector and S. avermitilis panD version r1 were joined in aligation reaction (20 μL) containing 1× Quick ligation buffer (NewEngland Biolabs), 2 μL XbaI/PacI pMhCt089 vector, 2 μL XbaI/PacI S.avermitilis panD version r1 insert, and 1 μL Quick T4 DNA ligase (NewEngland Biolabs). The ligation reaction was incubated for 5 min at roomtemperature, and then placed on ice. 5 μL of this reaction was used totransform ONE SHOT® TOP10 chemically competent E. coli cells(Invitrogen) according to the manufacturer's instructions. Transformantswere plated onto 2× YT+amp plates and incubated at room temperature forthree days. Several of the resulting transformants were screened forproper insertion of the desired PCR products by XbaI and PacI digestion.A clone yielding the desired band sizes was confirmed to be correct byDNA sequencing and designated pMhCt092.

The final cloning step for the left-hand construct was to replace theald5680 5′ homology region present in pMhCt092 with the adh1202 5′homology region. A PCR product containing the sequence 5′ of the adh1202ORF, along with the desired additional restriction sites and flankingDNA for cloning was amplified with the primers 0612470 and 0612471. ThePCR reaction (50 μL) contained 1 μL of a 1 to 50 dilution of pGMEr140mini-prep plasmid DNA (a derivative of pMIBa107 described herein whereinthe PCR amplified region is identical), 1× iProof™ HF buffer (Bio-RadLaboratories), 100 pmol each of primers 0612470 and 0612471, 200 μM eachof dATP, dCTP, dGTP, and dTTP, and 1 unit of iProof™ High Fidelity DNApolymerase (Bio-Rad Laboratories). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 98° C. for 30 seconds followed by 34 cycles each at 98° C. for 10seconds, 59° C. for 20 seconds, and 72° C. for 30 seconds, with a finalextension at 72° C. for 10 minutes. Following thermocycling, the PCRreaction products were separated by 1% agarose gel electrophoresis inTAE buffer where an approximately 790 bp PCR product was excised fromthe gel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions.

To create a recipient vector for the above PCR product, the plasmidpMhCt092 was digested with HpaI and NotI, treated with 10 units calfintestinal phosphatase (New England Biolabs) at 37° C. for 30 minutes,and purified by 0.9% agarose gel electrophoresis in TAE buffer, and anapproximately 7.0 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The PCR product and linear vector were joined using an IN-FUSION™Advantage PCR Cloning Kit (Clontech) in a total reaction volume of 10 μLcomposed of 120 ng pMhCt092 vector digested with HpaI and NotI, 30 ng ofthe adh1202 5′ homology containing PCR product, 1× In-Fusion reactionbuffer (Clontech) and 1 μL of IN-FUSION™ enzyme (Clontech). The reactionwas incubated at 37° C. for 15 minutes, 50° C. for 15 minutes, and thenplaced on ice. The reaction was diluted with 40 μL of TE buffer and 2.5μL was used to transform SoloPack Gold SuperCompetent Cells (AgilentTechnologies) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion of the desired PCR products by digestion with BamHI andPstI. A clone yielding the desired band sizes was confirmed to becorrect by DNA sequencing and designated pMhCt095 (FIG. 12).

Plasmid pMhCt095 is a left-hand I. orientalis adh1202 targetingconstruct containing the PDC promoter driving expression of a S.avermitilis ADC gene codon-optimized for expression in I. orientalis(SEQ ID NO: 130), the TAL terminator, the ENO1 promoter drivingexpression of a second S. avermitilis ADC gene codon-optimized forexpression in I. orientalis (SEQ ID NO: 145), the I. orientalis RKIterminator, the I. orientalis URA3 promoter and the 5′ fragment of theI. orientalis URA3 ORF.

Example 3A-8: Right-Hand Fragments of Insertion Vectors with MultipleNucleotide Sequences for Expressing Aspartate 1-Decarboxylase (ADC) atthe Adh1202 Locus

A PCR product containing the 3′ fragment of the I. orientalis URA3 ORF,along with the desired additional restriction sites and flanking DNA forcloning was amplified with primers 0612275 and 0612276. The PCR reaction(50 μL) contained 1 μL of a 1 to 50 dilution of pMhCt069 mini-prepplasmid DNA (supra), 1× iProof™ HF buffer (Bio-Rad Laboratories), 100pmol each of primers 0612275 and 0612276, 200 μM each of dATP, dCTP,dGTP, and dTTP, and 1 unit of iProof™ High Fidelity DNA polymerase(Bio-Rad Laboratories). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific) programmed for one cycle at 98° C.for 30 seconds followed by 32 cycles each at 98° C. for 10 seconds, 59°C. for 20 seconds, and 72° C. for 45 seconds, with a final extension at72° C. for 10 minutes. Following thermocycling, the PCR reactionproducts were separated by 1% agarose gel electrophoresis in TAE bufferwhere an approximately 1155 bp PCR product was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturers instructions.

A fragment containing the TDH3 promoter, XbaI and PacI restriction sitesfor insertion of an ectopic gene, and the PDC terminator was excisedfrom pMhCt069 via digestion with NotI and PmeI. 10 μg of a midi-prep ofpMhCt069 was digested with NotI and PmeI and then purified by 0.9%agarose gel electrophoresis in TAE buffer, and an approximately 1.85 kbpband was excised from the gel and purified using a NUCLEOSPIN® ExtractII Kit (Macherey-Nagel) according to the manufacturer's instructions.

A PCR product containing the sequence 3′ of the ald5680 ORF, along withthe desired additional restriction sites and flanking DNA for cloningwas amplified with the primers 0612277 and 0612278. The PCR reaction (50μL) contained 1 μL of a 1 to 50 dilution of pHJJ75 mini-prep plasmid DNA(FIG. 23), 1× iProof™ HF buffer (Bio-Rad Laboratories), 100 pmol each ofprimers 0612277 and 0612278, 200 μM each of dATP, dCTP, dGTP, and dTTP,and 1 unit of iProof™ High Fidelity DNA polymerase (Bio-RadLaboratories). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 32 cycles each at 98° C. for 10 seconds, 59° C. for 20seconds, and 72° C. for 45 seconds, with a final extension at 72° C. for10 minutes. Following thermocycling, the PCR reaction products wereseparated by 1% agarose gel electrophoresis in TAE buffer where anapproximately 844 bp PCR product was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The purified 1155 bp, 1.85 kbp, and 844 bp DNA fragments from above werethen inserted into pUC19 digested with EcoRI and HindIII using anIN-FUSION™ Advantage PCR Cloning Kit (Clontech) in a total reactionvolume of 10 μL composed of 150 ng of the fragment from the pUC19 vectordigested with HindIII and EcoRI; 66 ng of the 1155 bp DNA containing the3′ portion of the URA3 split marker; 106 ng of the 1.85 kbp fragmentdigested with PmeI and NotI and containing the TDH3 promoter, XbaI andPacI restriction sites for insertion of an ectopic gene, and PDCterminator from pMhCt069; 48 ng of the 844 bp PCR product containing the3′ ald5680 flanking DNA; 1× In-Fusion reaction buffer (Clontech) and 1μL of IN-FUSION™ enzyme (Clontech). The reaction was incubated at 37° C.for 15 minutes, 50° C. for 15 minutes, and then placed on ice. Thereaction was diluted with 40 μL of TE buffer and 2.5 μL was used totransform SoloPack Gold SuperCompetent Cells (Agilent Technologies)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at room temperature for three days.Several of the resulting transformants were screened for properinsertion of the desired PCR products by SalI and HpaI digestion. Aclone yielding the desired band sizes was confirmed to be correct by DNAsequencing and designated ald5680 right #20.

The TKL terminator, the PGK1 promoter, XbaI and PacI restriction sites,and a shorter version of the PDC terminator region was added between theTDH3 promoter and 3′ ald5680 flanking DNA of ald5680 right #20 asfollows. The TKL terminator along with the desired additionalrestriction sites and flanking DNA for cloning was amplified with theprimers 0612356 and 0612357. The desired PCR product was amplified usinga temperature gradient for the annealing temperature and DMSO in somereactions. Four identical PCR reactions were prepared, with each PCRreaction (50 μL) containing 1 μL of a 1 to 50 dilution of pACN23mini-prep plasmid DNA (FIG. 20), 1× iProof™ HF buffer (Bio-RadLaboratories), 100 pmol each of primers 0612356 and 0612357, 200 μM eachof dATP, dCTP, dGTP, and dTTP, and 1 unit of iProof™ High Fidelity DNApolymerase (Bio-Rad Laboratories). A second set of four tubes was set upas above except that the reactions each included the addition of 1.5 μLof DMSO. The PCRs were performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 32 cycles each at 98° C. for 10 seconds, X° C. for 20seconds, where X=47.6° C., 51.8° C., 57.1° C., or 62.1° C., and 72° C.for 45 seconds, with a final extension at 72° C. for 10 minutes. A PCRwith and without DMSO was run for each annealing temperature shown.Following thermocycling, 10 μL of each PCR reaction was separated by 1%agarose gel electrophoresis in TAE buffer. Visualization of this gelrevealed that PCR reactions performed with DMSO at the two highestannealing temperatures and without DMSO at the two lowest annealingtemperature gave the highest yield of the desired 844 bp product. Thesefour PCRs were combined, separated by 1% agarose gel electrophoresis inTAE buffer, where the approximately 844 base pair PCR product wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

PCR amplification of the desired PGK1 promoter region was done as a twostep process. First, a PCR product containing the PGK1 promoter DNA wascloned following amplification with the following primers 0612150 and0612151. The PCR reaction (50 μL) contained 3 μL of pJLJ49 mini-prep DNA(FIG. 25), 1× Pfx amplification buffer (Invitrogen), 2 mm MgSO₄, 100pmol each of primers 0612150 and 0612151, 200 μM each of dATP, dCTP,dGTP, and dTTP, and 1.25 Units Platinum® Pfx DNA polymerase(Invitrogen). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 95° C. for 2 minutesfollowed by 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute,and 72° C. for 3 minutes, with a final extension at 72° C. for 10minutes. Following thermocycling, the PCR reaction products wereseparated by 1% agarose gel electrophoresis in TAE buffer where anapproximately 630 bp PCR product was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The approximately 630 bp PCR product was cloned into pCR4™BLUNT TOPO®(Invitrogen) vector using the Zero Blunt® TOPO® PCR cloning kit forsequencing (Invitrogen) according to the manufacturer's instructions. Ina total reaction volume of 6 μL either 0.5 or 4 μL of the 630 bp PCRproduct, 1 μL salt solution (Invitrogen) and 1 μL pCR4™BLUNT TOPO®(Invitrogen) were incubated together at room temperature for 15 minutes.2 μL of each cloning reaction was transformed into One Shot® TOP10Chemically Competent E. coli (Invitrogen) cells according tomanufacturer's instructions. Transformants were plated onto LB+kanplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired PCRproduct by EcoRI digestion. A clone yielding the desired band sizes wasconfirmed to be correct by DNA sequencing and designated PGK1_in_TOPO.

The PGK1 promoter from PGK1_in_TOPO was isolated and purified prior touse as a PCR template as follows. 25 μL of a midi-prep of PGK1_in_TOPOwas digested with XbaI and PacI and purified by 0.9% agarose gelelectrophoresis in TAE buffer, and an approximately 640 bp band wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

The PGK1 promoter along with the desired additional restriction sitesand flanking DNA for cloning was amplified with the primers 0612358 and0612359 using a temperature gradient. Eight identical PCR reactions wereset up, each PCR reaction (50 μL) contained 20 ng of purified PGK1promoter DNA via XbaI and PacI digestion of PGK1_in_TOPO, 1× Herculasereaction buffer (Agilent Technologies), 100 pmol each of primers 0612358and 0612359, 200 μM each of dATP, dCTP, dGTP, and dTTP, and 2.5 units ofHerculase HotStart DNA Polymerase (Agilent Technologies). The PCRs wereperformed in an EPPENDORF® MASTERCYCLER® (Eppendorf Scientific)programmed for one cycle at 98° C. for 30 seconds followed by 32 cycleseach at 98° C. for 10 seconds, X° C. for 20 seconds, where X=53.7° C.,55.4° C., 57.6° C., 60.0° C., 62.4° C., 64.8° C., 66.9° C., 68.6° C.,and 72° C. for 45 seconds, with a final extension at 72° C. for 10minutes. Following thermocycling, 10 μL of each PCR reaction wasseparated by 1% agarose gel electrophoresis in TAE buffer. Visualizationof this gel revealed that four PCR reactions performed with the highestannealing temperature gave the highest yield of the desiredapproximately 700 bp product. These four PCRs were combined, separatedby 1% agarose gel electrophoresis in TAE buffer, where the approximately700 base pair PCR product was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

Plasmid ald5680_right #20 contains approximately 870 bp of the regiondownstream from the I. orientalis PDC ORF as the PDC terminator region.However, this region is likely larger than necessary for proper functionas a terminator and if maintained in its current size might serve as acatalyst for unwanted homologous recombination to the PDC locus.Therefore, a PCR product to replace the PDC terminator in ald5680_right#20 with a smaller version was amplified with the primers 0612360 and0612361. The desired PCR product was amplified using a temperaturegradient for the annealing temperature and DMSO in some reactions. Fouridentical PCR reactions were set up, each PCR reaction (50 μL) contained1 μL of a 1 to 50 dilution of pJLJ49 mini-prep plasmid DNA (FIG. 25), 1×iProof™ HF buffer (Bio-Rad Laboratories), 100 pmol each of primers0612360 and 0612361, 200 μM each of dATP, dCTP, dGTP, and dTTP, and 1unit of iProof™ High Fidelity DNA polymerase (Bio-Rad Laboratories). Asecond set of four tubes was set up as above except that the reactionseach included the addition of 1.5 μL of DMSO. The PCRs were performed inan EPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for onecycle at 98° C. for 30 seconds followed by 32 cycles each at 98° C. for10 seconds, X° C. for 20 seconds, where X=47.6° C., 51.8° C., 57.1° C.,or 62.1° C., and 72° C. for 45 seconds, with a final extension at 72° C.for 10 minutes. A PCR with and without DMSO was run for each annealingtemperature shown. Following thermocycling, 10 μL of each PCR reactionwas separated by 1% agarose gel electrophoresis in TAE buffer.Visualization of this gel revealed that the four PCR reactions performedwith DMSO, regardless of annealing temperature, gave the highest yieldof the desired 338 bp product. These four PCRs were combined, separatedby 1% agarose gel electrophoresis in TAE buffer, where the approximately338 base pair PCR product was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

PCR was used to create a single amplification product fusingapproximately 700 bp PGK1 containing PCR product to the 338 bp PDCterminator product. The PCR reaction (50 μL) contained 107 ng of thePGK1 containing PCR product, 56 ng of the PDC terminator containing PCRproduct, 1× Phusion HF buffer (New England Biolabs), 100 pmol each ofprimers 0612358 and 0612361, 200 μM each of dATP, dCTP, dGTP, and dTTP,and 1 unit of Phusion™ High-Fidelity DNA polymerase (New EnglandBiolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 30 secondsfollowed by 34 cycles each at 98° C. for 10 seconds, 56° C. for 20seconds, and 72° C. for 2 minutes and 45 seconds, with a final extensionat 72° C. for 10 minutes. Following thermocycling, the PCR reactionproducts were separated by 0.9% agarose gel electrophoresis in TAEbuffer where an approximately 1020 base pair PCR product was excisedfrom the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

The plasmid ald5680_right #20 was digested with XbaI and NotI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and purified by 0.9% agarose gel electrophoresis inTAE buffer. A band of approximately 5.6 kbp was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

The purified 487 bp and 1020 bp PCR products from above were theninserted into the digested ald5680_right#20 fragment using an IN-FUSION™Advantage PCR Cloning Kit (Clontech) in a total reaction volume of 10 μLcomposed of 150 ng of the ald5680_right #20 vector digested with XbaIand NotI, 13 ng of the TKL terminator PCR product, 28 ng of the PGK1promoter-PDC terminator PCR product, 1× In-Fusion reaction buffer(Clontech) and 1 μL of IN-FUSION™ enzyme (Clontech). The reaction wasincubated at 37° C. for 15 minutes, 50° C. for 15 minutes, and thenplaced on ice. The reaction was diluted with 40 μL of TE buffer and 2.5μL was used to transform SoloPack Gold SuperCompetent Cells (AgilentTechnologies) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion of the desired PCR products by AccI digestion. A cloneyielding the desired band sizes was confirmed to be correct by DNAsequencing and designated pMhCt091.

Plasmid pMhCt091 is an empty right-hand I. orientalis ald5680 targetingconstruct containing the 3′ fragment of the I. orientalis URA3 ORF, theI. orientalis TDH3 promoter followed by NheI and AscI restriction sitesfor addition of a gene of interest, the I. orientalis TKL terminator,the I. orientalis PGK1 promoter followed by XbaI and PacI restrictionsites for addition of a second gene of interest, the I. orientalis PDCterminator, and flanking DNA to target homologous recombination to the3′ ald5680 locus.

S. avermitilis panD version r5 (SEQ ID NO: 146) was synthesized in thevector 1075328_SaPanD_r5 by GeneArt®. The XbaI and PacI restrictionsites were changed to the desired NheI and AscI sites for cloning intothe 5′ cloning site of pMhCt091 by PCR. The PCR reaction (50 μL)contained 1 μL of a 1 to 50 dilution of 1075328_SaPanD_r5 mini-prepplasmid DNA (GeneArt®), 1× iProof™ HF buffer (Bio-Rad Laboratories), 100pmol each of primers 0612378 and 0612379, 200 μM each of dATP, dCTP,dGTP, and dTTP, 1.5 μL DMSO and 1 unit of iProof™ High Fidelity DNApolymerase (Bio-Rad Laboratories). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 98° C. for 30 seconds followed by 34 cycles each at 98° C. for 10seconds, 59° C. for 20 seconds, and 72° C. for 30 seconds, with a finalextension at 72° C. for 10 minutes. Following thermocycling, the PCRreaction products were separated by 0.9% agarose gel electrophoresis inTAE buffer where an approximately 471 base pair PCR product was excisedfrom the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

The plasmid pMhCt091 (supra) was digested with NheI and AscI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs) at 37°C. for 30 minutes, and purified by 0.9% agarose gel electrophoresis inTAE buffer. A band of approximately 6.9 kbp was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions. The purified panD r5 containing PCRproduct from above was then inserted into the digested pMhCt091 fragmentusing an IN-FUSION™ Advantage PCR Cloning Kit (Clontech) in a totalreaction volume of 10 μL composed of 150 ng of the pMhCt091 vectordigested from NheI and AscI, 19 ng of the panD r5 PCR product, 1×In-Fusion reaction buffer (Clontech) and 1 μL of IN-FUSION™ enzyme(Clontech). The reaction was incubated at 37° C. for 15 minutes, 50° C.for 15 minutes, and then placed on ice. The reaction was diluted with 40μL of TE buffer and 2.5 μL was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired PCRproducts by SmaI digestion. A clone yielding the desired band sizes wasconfirmed to be correct by DNA sequencing and designated pMhCt093.

The plasmid pMhCtO93 was digested with XbaI and PacI, treated with 10units calf intestinal phosphatase (New England Biolabs) at 37° C. for 30minutes, and purified by 0.9% agarose gel electrophoresis in TAE buffer,and an approximately 7.4 kbp band was excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. S. avermitilis panD version r2 (SEQ ID NO:147) was codon-optimized for expression in I. orientalis and synthesizedin the vector pMA-T by GeneArt®. The plasmid was digested with XbaI andPacI and the resulting fragments were separated by 0.9% agarose gelelectrophoresis in TAE buffer. A band of approximately 434 bp wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

The purified ˜434 bp fragment above was cloned into the pMhCt093 vectordigested with XbaI and PacI in a ligation reaction (20 μL) containing 1×Quick ligation buffer (New England Biolabs), 2 μL of the pMhCt093 vectordigested with XbaI and PacI, 2 μL of the ˜434 bp fragment above, and 1μL Quick T4 DNA ligase (New England Biolabs). The ligation reaction wasincubated for 5 min at room temperature, and then the tube placed onice. 5 μL of this reaction was used to transform SoloPack GoldSuperCompetent Cells (Agilent Technologies) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at room temperature for three days. Several of theresulting transformants were screened for proper insertion of thedesired PCR products by digestion with XbaI and PacI. Isolate pMhCt094was chosen for future work.

The final cloning step for the right-hand construct was to replace theald5680 3′ homology region present in pMhCt094 with the adh1202 3′homology region. A PCR product containing the sequence just 3′ of theadh1202 ORF, along with the desired additional restriction sites andflanking DNA for cloning was amplified with the primers 612472 and612473. The PCR reaction (50 μL) contained 1 μL of a 1 to 50 dilution ofpGMEr140 (supra) mini-prep plasmid DNA, 1× iProof™ HF buffer (Bio-RadLaboratories), 100 pmol each of primers 0612472 and 0612473, 200 μM eachof dATP, dCTP, dGTP, and dTTP, and 1 unit of iProof™ High Fidelity DNApolymerase (Bio-Rad Laboratories). The PCR was performed in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for one cycleat 98° C. for 30 seconds followed by 34 cycles each at 98° C. for 10seconds, 59° C. for 20 seconds, and 72° C. for 30 seconds, with a finalextension at 72° C. for 10 minutes. Following thermocycling, the PCRreaction products were separated by 1% agarose gel electrophoresis inTAE buffer where an approximately 620 base pair PCR product was excisedfrom the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions.

To create a recipient vector for the above PCR product, the plasmidpMhCt094 was digested with SacII and NotI, treated with 10 units calfintestinal phosphatase (New England Biolabs) at 37° C. for 30 minutes,and purified by 0.9% agarose gel electrophoresis in TAE buffer, and anapproximately 7.0 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. The PCR product and linear vector werejoined using an IN-FUSION™ Advantage PCR Cloning Kit (Clontech) in atotal reaction volume of 10 μL composed of 191 ng of the pMhCt094 vectordigested with SacII and NotI, 36 ng of the adh1202 3′ homologycontaining PCR product, 1× In-Fusion reaction buffer (Clontech) and 1 μLof IN-FUSION™ enzyme (Clontech). The reaction was incubated at 37° C.for 15 minutes, 50° C. for 15 minutes, and then placed on ice. Thereaction was diluted with 40 μL of TE buffer and 2.5 μL was used totransform SoloPack Gold SuperCompetent Cells (Agilent Technologies)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion of thedesired PCR products by digestion with NsiI and PvuI. A clone yieldingthe desired band sizes was confirmed to be correct by DNA sequencing anddesignated pMhCt096 (FIG. 13).

Plasmid pMhCt096 is a right-hand I. orientalis adh1202 targetingconstruct containing the 3′ fragment of the I. orientalis URA3 ORF, theI. orientalis TDH3 promoter driving expression of a third S. avermitilisADC gene codon-optimized for expression in I. orientalis (SEQ ID NO:146), the I. orientalis TKL terminator, the I. orientalis PGK1 promoterdriving expression of a forth S. avermitilis ADC gene codon-optimizedfor expression in I. orientalis (SEQ ID NO: 147), the I. orientalis PDCterminator, and flanking DNA to target homologous recombination to the3′ adh1202 locus.

Example 3A-9: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC),β-Alanine Aminotransferase (BAAT), and 3-Hydroxypropionic AcidDehydrogenase (3-HPDH) at the Pdc Locus; and Aspartate 1-Decarboxylase(ADC) from Four Nucleotide Sequences at the Adh1202 Locus

Examples 3A-7 and 3A-8 above describe creation of left-hand andright-hand constructs for targeting expression of four nucleotidevariants of the S. avermitilis ADC gene codon-optimized for expressionin I. orientalis at the adh1202 locus. Prior to transformation into I.orientalis CNB1, 10 μg of pMhCt095 was digested with HpaI and SacII torelease the desired transforming DNA from the pUC19 backbone vector.Likewise, 10 μg of pMhCt096 was digested with EcoRI and SacII to releasethe desired transforming DNA from the pUC19 backbone vector. The ˜5 kbpexpression cassette containing band was separated from the pUC19backbone DNA by gel electrophoresis, excised from the gel, and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. 30 μL elution buffer was used for theelution step. An equimolar ratio of pMhCt095 and pMhCt096 lineartransformation DNA totaling 10 μL were used to transform strain yMhCt010(supra). Transformants were selected on ura selection plates and placedat 37° C. for growth. Approximately twelve transformants were picked thefollowing day and restreaked for single colonies to ura selection platesand grown at 37° C. overnight, and then a single colony was picked fromeach of the streaks generated by each initial transformant andrestreaked to ura selection plates. After another night of growth at 37°C., a final single colony was picked from each streak and restreaked toa ura selection plate and grown overnight at 37° C. After this secondround of single colony purification and outgrowth, genomic DNA wasprepared for use in PCR to verify the desired targeted integrationoccurred as described herein. Correct targeting of the pMhCt095 andpMhCt096 fragments to the adh1202 locus was verified using primers0611718 and 0611632 (supra). Primer 0611718 binds in the PDC terminatorregion present in pMhCt096, while primer 0611632 binds in adh1202 locusDNA 3′ of the region targeted and amplifies in the anti-sense direction.Generation of an approximately 727 bp band from PCRs with these primersindicated the occurrence of the desired integration event at the adh1202locus.

A PCR reaction (25 μL) contained 0.5 μL genomic DNA for the strain to bescreened, 1× Crimson Taq™ Reaction Buffer (New England Biolabs), 25 pmolof the sense primer, 25 pmol of the anti-sense primer, 200 μM each ofdATP, dCTP, dGTP, and dTTP, and 0.625 units of Crimson Taq™ DNApolymerase (New England Biolabs). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific) programmed for one cycle at 95° C.for 30 seconds followed by 30 cycles each at 95° C. for 30 seconds, 50°C. for 30 seconds, and 68° C. for 2.5 minutes, with a final extension at68° C. for 10 minutes. Following thermocycling, the PCR reactionproducts were separated by 1% agarose gel electrophoresis in TAE bufferand the sizes of the bands visualized and interpreted as describedabove. Two independently isolated transformants giving the desired 727bp band were designated yMhCt019 or 95/96 2 (see genotype in Table 21).

TABLE 21 Transformant genotype Strain Parent strain Genotype yMhCt019yMhCt010 adh1202Δ::(PDC_(promo)-Opt.SaPanD r10, ENO1_(promo)- 95/96 2Opt.SaPanD r1, URA3, TDH3_(promo)-Opt.SaPanD r5, PGK1_(promo)-Opt.SaPanDr2)/ADH1202 pdcΔ::(PDC_(promo)- Opt.SaPanD ENO1_(promo)-Opt.ScUGA1,URA3- Scar, TDH3_(promo)-Opt.ScYMR226C)/pdcΔ::(PDC_(promo)- Opt.SaPanD,ENO1_(promo)-Opt.ScUGA1, URA3-Scar, TDH3_(promo)-Opt.ScYMR226C)ura3-/ura3-

A ura-derivative of yMhCt019 then was isolated as described herein.Genomic DNAs from several FOA resistant colonies of yMhCt019 werescreened by PCR for the desired loop-out event with the primers 0611815and 0612795. Primer 0611815 anneals in the RKI terminator of theleft-hand construct and amplifies toward the URA3 promoter, while primer0612795 anneals within 3′ adh1202 homology (of pMhCt096 or endogenousadh1202 locus) back toward 5′ region. PCR reactions were carried out asdescribed for the isolation of yMhCt019 above except that the length ofthe extension phase was changed to 3.5 minutes. Generation of a 3.7 kbpband with these primers indicates that the desired loop-out event hasoccurred and only the URA3 promoter scar remains at the modified adh1202locus, while an approximately 5.1 kbp band would indicate the presenceof the intact URA3 promoter-URA3 ORF-URA3 terminator-URA3 promotercassette, indicating the desired recombination event did not occur. Astrain that gave the desired 3.7 kbp band was kept and designatedyMhCt021.

In order to isolate a derivative of yMhCt021 homozygous for the multiplepanD expression cassette at adh1202, yMhCt021 was transformed withlinearized pMhCt095 and pMhCt096 as described above. After two rounds ofsingle colony purification and outgrowth, genomic DNA was prepared foruse in PCR to verify the desired targeted integration occurred. Correcttargeting of the pMhCt095 and pMhCt096 fragments to the remainingwild-type adh1202 locus of yMhCt021 was verified with the primers0612891 and 0612893. Primer 0612891 anneals in the 3′ region of SaPanDr1 plus half of the PacI site after r1 stop of pMhCt095. Primer 0612893anneals in the extreme 5′ region of SaPanD r5, includes the NheI siteand leader of pMhCt096, and amplifies in reverse complementarydirection.

Generation of a 3.2 kbp band with these primers indicates the presenceof an intact URA3 promoter-URA3 ORF-URA3 terminator-URA3 promotercassette as expected from the second integration event via pMhCt095 andpMhCt096 on the remaining adh1202 wild-type locus of yMhCt021, while anapproximately 1.7 kbp band would indicate the presence of the URA3 scarsite at the other adh1202 locus (from the initial integration event andsubsequent URA3 marker loop-out). PCR reactions were carried out asdescribed for the isolation of yMhCt019 above except that the length ofthe extension phase was changed to 3.5 minutes. A strain that gave bothof these band sizes was designated yMhCt022 (see genotype in Table 22).

TABLE 22 Transformant genotype Strain Parent strain Genotype yMhCt022yMhCt021 adh1202Δ::(PDC_(promo)-Opt.SaPanD r10, ENO1_(promo)- Opt.SaPanDr1, URA3, TDH3_(promo)-Opt.SaPanD r5, PGK1_(promo)-Opt.SaPanDr2)/adh1202Δ::(PDC_(promo)- Opt.SaPanD r10, ENO1_(promo)-Opt.SaPanD r1,URA3-Scar, TDH3_(promo)-Opt.SaPanD r5, PGK1_(promo)-Opt.SaPanD r2)pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)-Opt.ScUGA1, URA3-Scar,TDH3_(promo)-Opt.ScYMR226C)/pdcΔ::(PDC_(promo)- Opt.SaPanD,ENO1_(promo)-Opt.ScUGA1, URA3-Scar, TDH3_(promo)-Opt.ScYMR226C)ura3-/ura3-

Strains were grown in shake flasks and CFE were prepared and assayed foraspartate decarboxylase (ADC) activity as herein. The experimentalresults are shown in Table 23A.

TABLE 23A Transformant enzyme activity data Gene Strain OverexpressedADC activity MBin500 N/A 0.00 (control) yMhCt019 ADC (SEQ ID NOs: 130,145, 146, and 147), 2.18 95/96 2 gabT (SEQ ID NO: 141), 2.52 3-HPDH (SEQID NO: 144)

The aspartate 1-decarboxylase (ADC), beta-alanine aminotransferase(BAAT) and 3HP dehydrogenase (3-HPDH) activities in CFE prepared fromstrains MBin500 (control), yMhCt019, 95/96-2, yMhCt008 and yMhCt022 werecompared. The results of this experiment are shown in Table 23B.

TABLE 23B Transformant enzyme activity data Gene ADC BAAT 3HP DH StrainGenes Overexpressed Sources Activity Activity Activity MBin500 N/A N/A0.002 0.61 0.4 (control) yMhCt019 YMR226c (SEQ ID NO: 144) S. cerevisiae0.789 14.53 72.0 ADC (SEQ ID NOs: 130, 145, S. avermitilis 46 and 147)UGA1 (SEQ ID NO: 141) S. cerevisiae 95/96-2 YMR226c (SEQ ID NO: 144) S.cerevisiae 0.891 20.42 73.2 ADC (SEQ ID NOs: 130, 145, S. avermitilis146 and 147) UGA1 (SEQ ID NO: 141) S. cerevisiae yMhCt008 YMR226c (SEQID NO: 144) S. cerevisiae 0.272 13.14 61.1 ADC (SEQ ID NO: 130) S.avermitilis UGA1 (SEQ ID NO: 141) S. cerevisiae yMhCt022 YMR226c (SEQ IDNO: 144) S. cerevisiae 1.233 15.44 66.7 ADC (SEQ ID NOs: 130, 145, S.avermitilis 146 and 147) UGA1 (SEQ ID NO: 141) S. cerevisiae

Strains yMhCt019 and 95/96 2 were also analyzed by SDS-PAGE as describedherein. Both strains showed a protein band at 53 kD, 29 kD, ˜14 kD, andat ˜3 kD. The sizes of the 53 kD and 29 kD protein bands are consistentwith the sizes of the proteins encoded by the UGA1 and YMR226c genes,respectively. The combined sizes of the 14 and 3 kD protein bands areconsistent with the post-translationally cleaved protein encoded by thepanD gene. The 53 kD, 29 kD, 14 kD and 3 kD proteins were not observedin the SDS-PAGE analysis of the control strain MBin500.

Strains MBin500 and yMhCt019 were evaluated in bioreactors for 3-HPproduction, using the method described herein. Control strain MBin500produced no detectable 3-HP (average of two independent fermentations).Strain yMhCt019 produced 5.23 g/L 3-HP (average of three independentfermentations).

Example 3A-10: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)from Four Nucleotide Sequences at the Adh1202 Locus

Additional constructs were designed to incorporate four copies ofnucleotides encoding an alternate ADC from B. licheniformis (SEQ ID NO:139) at the adh1202 locus. In a similar approach to that describedabove, a left-hand and a right-hand construct were designed to allowhomologous recombination at the I. orientalis adh1202 locus.

Construction of a Left-Hand Fragment

The plasmid pMhCt095 (supra) was digested with XbaI and PacI andpurified by 1% agarose gel electrophoresis in TBE buffer as describedherein. A band at approximately 7.3 kbp was excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto the manufacturer's instructions.

A Bacillus licheniformis aspartate decarboxylase (ADC) panD gene wascodon-optimized for expression in I. orientalis (version 1; SEQ ID NO:149) and synthetically constructed into plasmid 1110206 (GeneArt®).Plasmid 1110206 was digested with XbaI and PacI and purified by 1%agarose gel electrophoresis in TBE buffer as described herein. A band atapproximately 380 bp was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The ˜380 bp purified fragment was ligated into the 7.3 kbp pMhCt095linearized vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 60.5 ng of the digested pMhCt095,6.3 ng of the 380 bp fragment from 1110206, 1 μL 10× ligation bufferwith 10 mM ATP (New England Biolabs), and 1 μL T4 ligase (New EnglandBiolabs). The reaction was incubated for 1.5 hr at room temperature anda 3 μL aliquot of the reaction was transformed into ONE SHOT® TOP10chemically competent E. coli cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated on 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by restriction digestusing XbaI and PacI. A clone yielding the correct digested band size wasdesignated pMeJi309.

The plasmid pMeJi309 was digested with NheI and AscI and purified by 1%agarose gel electrophoresis in TBE buffer. A band of approximately 7.3kbp was excised from the gel and purified using a NUCLEOSPIN® Extract IIKit (Macherey-Nagel) according to the manufacturer's instructions.

The Bacillus licheniformis aspartate decarboxylase panD gene was againcodon-optimized for expression in I. orientalis (version 2; SEQ ID NO:148) and synthetically constructed into plasmid 1110205 (GeneArt®). APCR was performed on a mixture containing 3 μL 1110205, 25 μM each ofprimers 0612695 and 0612724, 1× pfx amplification buffer (Invitrogen), 2mm MgSO₄, 1.25 Units Platinum® pfx DNA polymerase (Invitrogen) in afinal volume of 50 μL. The amplification reactions were incubated in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for 1 cycleat 95° C. for 2 minutes; 25 cycles each at 95° C. for 1 minute, 55° C.for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 3minutes.

The PCR product from the amplification reactions was purified by 1%agarose gel electrophoresis in TBE buffer. The approximately 400 bp bandwas excised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to manufacturer's instructions. The purifiedPCR product was inserted into the digested pMeJi309 vector above usingan IN-FUSION™ Advantage PCR Cloning Kit (Clontech) in a reactioncontaining 93 ng pMeJi309 vector fragment, 52 ng PCR product above, 2 μL1× IN-FUSION™ reaction buffer (Clontech), and 1 μL of IN-FUSION™ enzyme(Clontech). The reaction was incubated at 37° C. for 15 minutes, 50° C.for 15 minutes, and then placed on ice. A 2.5 μL sample of the reactionwas transformed into ONE SHOT® TOP10 chemically competent E. coli cells(Invitrogen) according to manufacturer's instructions. Transformantswere plated on 2× YT+amp plates and incubated at 37° C. overnight.Several of the resulting transformants were screened for properinsertion by restriction digest using XbaI and PacI. A clone yieldingthe correct digested band size was confirmed to be correct by DNAsequencing and designated pMeJi310-2 (FIG. 14).

Construction of a Right-Hand Fragment

The plasmid pMhCt096 (supra) was digested with XbaI and PacI andpurified by 1% agarose gel electrophoresis in TBE buffer. A band atapproximately 4.8 kbp was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The Bacillus licheniformis aspartate decarboxylase panD gene was againcodon-optimized for expression in I. orientalis (version 3; SEQ ID NO:151) and synthetically constructed into plasmid 1110208 (GeneArt®).Plasmid 1110208 was digested with XbaI and PacI and purified by 1%agarose gel electrophoresis in TBE buffer, and an approximately 380 bpband was excised from the gel and purified using a NUCLEOSPIN® ExtractII Kit (Macherey-Nagel) according to the manufacturer's instructions.

The 380 bp fragment above was ligated into the 7.3 kbp pMhCt096linearized vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 72.2 ng digested pMhCt096, 6.9 ng380 bp fragment from 1110208, 1 μL 10× ligation buffer with 10 mM ATP,and 1 μL T4 ligase. The reaction was incubated for 1 and a half hours atroom temperature and a 3 μL aliquot of the reaction was transformed intoONE SHOT® TOP10 chemically competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated on2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion byrestriction digest using nheI and ascI. A done yielding correct digestedband size was designated pMeJi311.

The plasmid pMeJi311 was digested with enzymes NheI and AscI andpurified by 1% agarose gel electrophoresis in TBE buffer, and anapproximately 7.3 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The Bacillus licheniformis aspartate decarboxylase panD gene was againcodon-optimized for expression in I. orientalis (version 4; SEQ ID NO:150) and synthetically constructed into plasmid 1110207 (GeneArt®). APCR was performed on a mixture containing 3 μL 1110207, 25 μM each of0612698 and 0612725, 1× pfx amplification buffer (Invitrogen), 2 mMMgSO₄, 1.25 units Platinum® pfx DNA polymerase (Invitrogen) in a finalvolume of 50 μL. The amplification reaction was incubated in anEPPENDORF® MASTERCYCLER® (Eppendorf Scientific) programmed for 1 cycleat 95° C. for 2 minutes; 25 cycles each at 95° C. for 1 minute, 55C for1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 3 minutes.

The PCR product from the amplification reaction was purified by 1%agarose gel electrophoresis in TBE buffer. A band at approximately 400bp was excised from the gel and purified using a NUCLEOSPIN® Extract IIKit (Macherey-Nagel) according to manufacturer's instructions. Thepurified PCR product was inserted into the digested pMeJi311 vectorabove using an IN-FUSION™ Advantage PCR Cloning Kit (Clontech) in areaction containing 65.1 ng of the pMeJi311 NheI to AscI digested vectorfragment, 85 ng of the PCR product above, 2 μL 1× IN-FUSION™ reactionbuffer (Clontech), and 1 μL of IN-FUSION™ enzyme (Clontech). Thereaction was incubated at 37° C. for 15 minutes, 50° C. for 15 minutes,and then placed on ice. A 2.5 μL sample of the reaction was transformedinto ONE SHOT® TOP10 chemically competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated on2× YT+amp plates and incubated for two days at room temperature. Severalof the resulting transformants were screened for proper insertion byrestriction digest using NheI and AscI. A clone yielding the correctdigested band size was confirmed to be correct by DNA sequencing anddesignated pMeJi312-2 (FIG. 15).

Integration of Left-Hand and Right-Hand Fragments

Plasmid pMeJi310-2 was digested with HpaI and Sac II and plasmidpMeJi312-2 was digested with EcoRI and SacII as described herein. Thesewere purified by 1% agarose gel electrophoresis in TBE buffer, and thetwo approximately 5 kbp bands were excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

I. orientalis CNB1 was transformed with the digested pMeJi310-2 andpMeJi312-2 DNA and correct locus targeting and transformation wasverified by Crimson Taq (New England Biolabs) PCR as described inherein. Primers 0612794 and 0611245 yielded an approximately 3.17 kbpband; primers 612479 and 0611632 yielded an approximately 1.48 kbp band;and primers 611248 and 612795 yielded an approximately 2.3 kbp band. Astrain which gave the expected bands for proper integration of theexpression cassette was designated MeJi409-2. A ura-derivative of strainMeJi409-2 was then obtained as described above.

Strains MBin500 and MeJi409-2 were evaluated in fermentation bioreactorsfor 3-HP production, using the method described herein. Control strainMBin500 produced no detectable 3-HP (average of two independentfermentations). Strain MeJi409-2 (one fermentation) produced 4.62 g/L.In order to account for differences in the amount of cell mass in thesefermentations compared to other (e.g., future) fermentations, the 3-HPconcentration per unit of cell mass (expressed as [g/L 3-HP]/[g/L drycell weight]) was calculated to be 0.20 for MeJi409-2.

Example 3A-11: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)and Aspartate Aminotransferase (AAT) at the Adh9091 Locus

The nucleotide sequence (SEQ ID NO: 13) that encodes the I. orientalisaspartate aminotransferase (AAT) of SEQ ID NO: 14 was PCR amplified fromI. orientalis genomic DNA using the primers 0611268 and 0611269. The PCRreaction (50 μL) contained 50 ng of strain I. orientalis genomic DNA, 1×Phusion HF buffer (New England Biolabs), 50 pmol each of primers 0611268and 0611269, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μL of 100%DMSO (New England Biolabs) and 1 unit of Phusion High Fidelity DNApolymerase (New England Biolabs). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific) programmed for one cycle at 98° C.for 2 minutes followed by 35 cycles each at 98° C. for 30 seconds, 55°C. for 30 seconds, and 72° C. for 1 minute and 30 seconds, with a finalextension at 72° C. for 7 minutes. Following thermocycling, the PCRreaction products were separated by 0.8% agarose gel electrophoresis inTBE buffer where an approximately 1278 bp PCR product was excised fromthe gel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen)according to the manufacturer's instructions. The total length of theresulting PCR fragment was approximately 1278 bp, with an NruIrestriction site at the 5′ end of the fragment and a PacI restrictionsite at its 3′ end.

The resulting 1278 bp fragment above comprising the AAT gene CDS (SEQ IDNO: 13) was then cloned into pCR2.1-TOPO vector and transformed intoOne-Shot TOP10 E. coli cells (Invitrogen) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired fragmentby Barn HI digestion. A clone yielding the desired band sizes wasconfirmed by sequencing and designated pGMEr11.

Plasmids pGMEr121 and pGMEr111 were double-digested with restrictionenzymes PacI and NruI. The resulting 7695 bp vector fragment, fromplasmid pGMEr121, and the resulting 1272 bp insert fragment comprisingthe AAT coding sequence, from plasmid pGMEr111, were separated by 0.8%agarose gel electrophoresis in 1×TBE buffer, excised from the gel, andpurified using the QIAQUICK® Gel Extraction Kit (Qiagen) according tothe manufacturer's instructions.

A ligation reaction was then set up with 3 μL of vector fragment, 4 μLof insert fragment, 2 μL of sterile dd water, 10 μL of 2× Quick LigaseBuffer and 1 μL of Quick T4 Ligase (Quick Ligation Kit, New EnglandBiolabs) and performed according to the manufacturer's instructions. A 5μL aliquot of the ligation reaction above was transformed intoXL10-Gold® Ultracompetent E. coli cells (Agilent Technologies) accordingto the manufacturer's instructions. Transformants were plated onto 2×YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion of thedesired insert by SmaI/PpuMI double digestion. A clone yielding thedesired band sizes was confirmed by sequencing and designated pGMEr126(FIG. 16).

Plasmids pGMEr126 comprises the I. orientalis AAT expression cassette,in which the gene transcription is controlled by the I. orientalis TDH3promoter and the TKL terminator, flanked by the truncated 3′ region ofthe URA3 coding sequence and the URA3 promoter, upstream; and by the 3′homology region with the I. orientalis adh9091 locus, downstream.

The S. avermitilis panD gene codon-optimized for expression in I.orientalis (SEQ ID NO: 130) was PCR amplified from the pMA-T vectorreceived from GeneArt® using the primers 061166 and 0611662. The PCRreaction (50 μL) contained 50 ng of strain plasmid DNA, 1× Phusion HFbuffer (New England Biolabs), 50 pmol each of primers 0611661 and0611662, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μL of 100% DMSO(New England Biolabs) and 1 unit of Phusion High Fidelity DNA polymerase(New England Biolabs). The PCR was performed in an EPPENDORF®MASTERCYCLER® (Eppendorf Scientific) programmed for one cycle at 98° C.for 2 minutes followed by 35 cycles each at 98° C. for 30 seconds, 55°C. for 30 seconds, and 72° C. for 30 seconds, with a final extension at72° C. for 7 minutes. Following thermocycling, the PCR reaction productswere separated by 0.8% agarose gel electrophoresis in TBE buffer wherean approximately 453 bp PCR product was excised from the gel andpurified using a QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions. The total length of the resulting PCRfragment was approximately 453 bp with an NruI restriction site at the5′ end of the fragment and an ApaI restriction site at the 3′ end.

The resulting 453 bp fragment, comprising the codon-optimized version ofS. avermitilis panD gene, was cloned into pCR2.1-TOPO vector andtransformed into One-Shot TOP10 E. coli cells (Invitrogen) according tothe manufacturer's instructions. Transformants were plated onto 2×YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion of thedesired fragment by EcoRI digestion. A plasmid yielding the desired bandsizes was confirmed by sequencing and designated pGMEr127.

Plasmids pGMEr127 and pGMEr125(a) were digested with restriction enzymesNruI and Apa I. Before stopping the digestion reactions 1 μL of CalfIntestinal Alkaline Phosphatase (New England Biolabs) was added to thepGMEr125(a) digestion in order to de-phosphorylate the ends and preventself-ligation. The resulting 8188 bp vector fragment, from plasmidpGMEr125(a) (supra), and the 440 bp insert fragment, comprising thecodon-optimized version of the S. avermitilis panD gene (SEQ ID NO: 130)from plasmid pGMEr127 (supra), were separated by 0.8% agarose gelelectrophoresis in 1×TBE buffer, excised from the gel and purified usinga QIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions.

A ligation reaction was then set up with 4 μL of vector fragment, 4 μLof insert fragment, 9 μL of 2× Quick Ligase Buffer and 1 μL of Quick T4Ligase (New England Biolabs) and performed according to themanufacturer's instructions. A 5 μL aliquot of the ligation reactionabove was transformed into XL10-Gold® Ultracompetent E. coli cells(Stratagene) according to the manufacturer's instructions. Transformantswere plated onto 2× YT+amp plates and incubated at 37° C. overnight.Several of the resulting transformants were screened for properinsertion of the desired insert by BamHI digestion. A clone yielding thedesired band sizes was confirmed and designated pGMEr130 (FIG. 17).

Plasmid pGMEr130 comprises a construct made of the following fragments:the 5′ flank of the I. orientalis adh9091 locus, an empty expressioncassette with the I. orientalis PDC promoter/TAL terminator, the panDexpression cassette (containing SEQ ID NO: 130) under control of the I.orientalis ENO1 promoter and the RKI terminator, and the truncated 5′fragment of the URA3 marker gene under control of the I. orientalis URA3promoter.

To determine whether yeast strain I. orientalis CNB1 was able to expressthe I. orientalis codon-optimized version of S. avermitilis panD gene(SEQ ID NO: 130) and the I. orientalis AAT gene (SEQ ID NO: 13), theexpression plasmids pGMEr130 and pGMEr126 were constructed. PlasmidpGMEr130 comprises (from 5′-3′) the 5′ flanking region for genomicintegration of the construct at the I. orientalis adh9091 locus, thepanD expression cassette under control by the ENO1 promoter and the RKIterminator, and the truncated 5′ portion of the URA3 selection markerdriven by the URA3 promoter. Plasmid pGMEr126 comprises (from 5′-3′) the3′ portion of the URA3 selection marker, the AAT gene expressioncassette under control by the TDH3 promoter and the TKL terminator, andthe 3′ flank for genomic integration of the construct at the I.orientalis adh9091 locus. All promoters and terminators were derivedfrom I. orientalis.

Plasmid pGMEr126 was digested with restriction enzyme EcoRI, whichexcised a 4758 bp fragment of interest, while plasmid pGMEr130 wasdigested with restriction enzyme Hind III creating a 5034 bp fragmentneeded for transformation. The 4758 bp and the 5034 bp fragments wereseparated by 0.8% agarose gel electrophoresis in 1×TBE buffer, excisedfrom the gel, and purified using the QIAQUICK® Gel Extraction Kit(Qiagen) according to the manufacturer's instructions.

I. orientalis CNB1 was cultured and co-transformed as described hereinwith approximately 500 ng of both the 4758 bp and 5034 bp linearfragments. Eight transformant strains were obtained and then cultured inshake flasks. The resulting broths were used to run an SDS-PAGE,Tris-HCl (Bio-Rad Laboratories) gel to detect the expression of the S.avermitilis panD gene codon-optimized for expression in I. orientalis(SEQ ID NO: 130) and of the I. orientalis AAT gene (SEQ ID NO: 13). Apositive strain was designated yGMEr008 and its broth was also used todetermine the ADC and the AAT activity levels as described above.

Example 3A-12: Yeast Strains Expressing Pyruvate Carboxylase (PYC),Aspartate 1-Decarboxylase (ADC) and Aspartate Aminotransferase (AAT) atthe Adh9091 Locus

The nucleotide sequence that encodes the I. orientalis pyruvatecarboxylase (PYC) of SEQ ID NO: 2 was PCR amplified from I. orientalisgenomic DNA using the primers 0611266 and 0611267. The PCR reaction (50μL) contained 50 ng of I. orientalis genomic DNA, 1× Phusion HF buffer(New England Biolabs), 50 pmol each of primers 0611266 and 0611267, 200μM each of dATP, dCTP, dGTP, and dTTP, 1.5 μL of 100% DMSO (New EnglandBiolabs) and 1 unit of Phusion High Fidelity DNA polymerase (New EnglandBiolabs). The PCR was performed in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific) programmed for one cycle at 98° C. for 2 minutesfollowed by 35 cycles each at 98° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute and 30 seconds, with a final extensionat 72° C. for 7 minutes. Following thermocycling, the PCR reactionproducts were separated by 0.8% agarose gel electrophoresis in TBEbuffer where an approximately 3557 bp PCR product was excised from thegel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen) accordingto the manufacturer's instructions. The resulting PCR fragment had anXbaI restriction site at the 5′ end of the fragment and a PacIrestriction site at its 3′ end.

The resulting 3557 bp fragment, comprising the I. orientalis PYC geneCDS (SEQ ID NO: 1), was cloned into pCR2.1-TOPO vector and transformedinto One-Shot TOP10 E. coli cells (Invitrogen) according to themanufacturer's instructions. Transformants were plated onto 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion of the desired fragmentby EcoRI digestion. Six clones yielding the desired band sizes wereconfirmed and designated pGMEr132.7, pGMEr132.14, pGMEr132.16,pGMEr132.25, pGMEr132.27 and pGMEr132.30. Sequencing analysis revealedthat plasmid pGMER132.14 has the proper PYC CDS but was missing the XbaIrestriction site at the CDS 5′ end. Since this restriction site isneeded to insert the PYC CDS in expression plasmid pGMEr125, the 315 bpHindIII fragment of plasmid pGMEr132.14 (comprising the 5′ end on thePYC CDS with the altered XbaI site) was replaced with the 315 bp HindIIIfragment from plasmid pGMEr132.7, which has a unaltered 5′ end of thePYC CDS including the correct XbaI site. The resulting 7173 bp HindIIIvector fragment, from plasmid pGMEr132.14, and the 315 bp HindIII insertfragment, from plasmid pGMEr132.7, were separated by 0.8% agarose gelelectrophoresis in 1×TBE buffer, excised from the gel, and purifiedusing a QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions.

A ligation reaction was then set up with 4 μL of vector fragment, 5 μLof insert fragment, 10 μL of 2× Quick Ligase Buffer and 1 μL of Quick T4Ligase (New England Biolabs) and performed according to themanufacturer's instructions. A 5 μL aliquot of the ligation reactionabove was transformed into One-Shot TOP10 E. coli cells (Invitrogen)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion andorientation of the desired insert by BamHI and XbaI double digestion. Aclone yielding the desired band sizes was confirmed and designatedpGMEr133.

In order to insert the I. orientalis PYC CDS downstream of the PDCpromoter in plasmid pGMEr125(b), plasmids pGMEr125(b) and pGMEr133(supra) were digested with PacI and XbaI. The resulting 8188 bp vectorfragment, from plasmid pGMEr125(b), and the 3553 bp insert fragment,comprising the I. orientalis PYC CDS (SEQ ID NO: 1) from plasmidpGMEr133, were separated by 0.8% agarose gel electrophoresis in 1×TBEbuffer, excised from the gel, and purified using the QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.

A ligation reaction was then set up with 3 μL of vector fragment, 6 μLof insert fragment, 10 μL of 2× Quick Ligase Buffer and 1 μL of Quick T4Ligase (New England Biolabs) and performed according to themanufacturer's instructions. A 5 μL aliquot of the ligation reactionabove was transformed into One-Shot TOP10 E. coli cells (Invitrogen)according to the manufacturer's instructions. Transformants were platedonto 2× YT+amp plates and incubated at 37° C. overnight Several of theresulting transformants were screened for proper insertion of thedesired fragment by BamHI digestion. A clone yielding the desired bandsizes was confirmed and designated pGMEr136.

Plasmid pGMEr136 comprises the 5′ flank of the I. orientalis adh9091locus, the I. orientalis PYC gene expression cassette (SEQ ID NO: 1)under control of the I. orientalis PDC promoter and TAL terminator, anempty expression cassette with an I. orientalis ENO1 promoter/RKIterminator, and the truncated 5′ fragment of the I. orientalis URA3marker gene under control of the URA3 promoter.

About 5 μg of plasmid pGMEr136 (supra) and 4 μg of plasmid pGMEr127 weredigested with restriction enzymes ApaI and Nru I. The resulting 11729 bpvector fragment, from plasmid pGMEr136, and the resulting insertfragment comprising the S. avermitilis panD gene codon-optimized forexpression in I. orientalis (SEQ ID NO: 130) (436 bp) from plasmidpGMEr127, were purified by 0.8% agarose gel electrophoresis in 1×TBEbuffer using a NucleoSpin® Extract II (Macherey-Nagel) according to themanufacturer's instructions.

A ligation reaction was then set up comprising 5 μL of vector fragment,4 μL of insert fragment, 9 μL of 2× Quick Ligase Buffer and 1 μL ofQuick T4 Ligase (New England Biolabs). The reaction was incubated atroom temperature for 1 hour. A 5 μL aliquot of the ligation reactionabove was transformed into ONE SHOT® TOP10 chemically competent E. colicells (Invitrogen) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened for thedesired insert by BamHI digestion. A clone yielding the desired bandsizes was chosen and designated pGMEr137 (FIG. 18).

Plasmid pGMEr137 comprises the I. orientalis PYC gene (SEQ ID NO: 1)under transcriptional control of the I. orientalis PDC promoter and TALterminator, the S. avermitilis panD gene codon-optimized for expressionin I. orientalis (SEQ ID NO: 130) under transcriptional control of theI. orientalis ENO1 promoter and RKI terminator, the URA3 promoterfollowed by the 5′ end of the URA3 marker and the 5′ flanking region ofthe I. orientalis adh9091 locus.

Plasmid pGMEr126 (supra) was digested with restriction enzyme EcoRI,which excised a 4758 bp fragment of interest; while plasmid pGMEr137(supra) was digested with restriction enzymes HpaI and NheI creating a8400 bp fragment. Both the 4758 bp and the 8400 bp fragments wereseparated by 0.8% agarose gel electrophoresis in 1×TBE buffer; the bandswere excised from the gel and purified using the NucleoSpin® Extract II(Macherey-Nagel) according to the manufacturer's instructions. I.orientalis CNB1 was cultured and co-transformed with approximately 500ng of both the 4758 bp and 8400 bp linear fragments as described herein,resulting in transformant yGMEr009.

Example 3A-13: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC),β-Alanine Aminotransferase (BAAT), and 3-Hydroxypropionic AcidDehydrogenase (3-HPDH) at the Pdc Locus; and Expressing PyruvateCarboxylase (PYC), Aspartate 1-Decarboxylase (ADC), and AspartateAminotransferase (AAT) at the Adh9091 Locus

To increase pyruvate carboxylase (PYC) and aspartate aminotransferase(AAT) activity in a strain already over-expressing an aspartateaminotransferase (AAT), a β-alanine aminotransferase (BAAT), and a 3-HPdehydrogenase (3-HPDH), strain yMhCt010 (supra) was transformed withlinear fragments of pGMEr137 (supra) and pGMEr126 (supra) as describedabove. After two rounds of single colony purification and outgrowth,genomic DNA was prepared for use in PCR to verify the desired targetedintegration occurred as described above. Correct targeting of thepGMEr137 and pGMEr126 fragments to the adh9091 locus was confirmed usingthe primers 0611814 and 0612055. Primer 0611814 anneals in the 3′ end ofthe TDH3 promoter of pGMEr126 and amplifies in the 3′ direction. Primer0612055 anneals 3′ of the adh9091 3′ flanking homology present inpGMEr126, so amplification of a PCR product with this primer pair willonly occur if the integration DNA targeted to the correct locus viahomologous recombination. The presence of an approximately 3066 bp bandfrom a PCR containing primers 0611814 and 0612055 indicates the desiredintegration of pGMEr126 and pGMEr137 fragments occurred at the adh9091locus.

After two rounds of single colony purification and outgrowth of severalindependent transformants of yMhCt010 with linear fragments of pGMEr137and pGMEr126, genomic DNA was prepared for use in PCR to verify thedesired targeted integration occurred as described above. Threeindependently isolated strains that gave an approximately 3066 bp bandfrom a PCR containing primers 0611814 and 0612055 were designatedyMhCt020, GMErin010#2, and GMErin010#3. These strains contain apolynucleotide (SEQ ID NO: 130) encoding the corresponding ADC (SEQ IDNO: 17) at both of the pdc loci and one of the adh9091 loci; apolynucleotide (SEQ ID NO: 141) encoding the corresponding gabT (SEQ IDNO: 24) at both of the pdc loci; a polynucleotide (SEQ ID NO: 144)encoding the corresponding 3-HPDH (SEQ ID NO: 129) at both of the pdcloci; a polynucleotide (SEQ ID NO: 1) encoding the corresponding PYC(SEQ ID NO: 2) at one of the adh9091 loci; and a polynucleotide (SEQ IDNO: 13) encoding the corresponding AAT (SEQ ID NO: 14) at one of theadh9091 loci (see Table 24).

TABLE 24 Transformant genotypes Strain Parent strain Genotype yMhCt020yMhCt010 adh9091Δ::(PDC_(promo)-pycCNB1, ENO1_(promo)- GMErin010 #2SaPanD(reverse), URA3, TDH3_(promo)-aat)/ADH9091 GMErin010 #3pdcΔ::(PDC_(promo)-Opt.SaPanD, ENO1_(promo)- Opt.ScUGA1, URA3-Scar,TDH3_(promo)- Opt.ScYMR226C)/pdcΔ::(PDC_(promo)-Opt.SaPanD,ENO1_(promo)-Opt.ScUGA1, URA3-Scar, TDH3_(promo)- Opt.ScYMR226C)ura3-/ura3-

Strains yMhCt020, GMErin010#2, and GMErin010#3 were grown in shakeflasks and CFEs were prepared and assayed for PYC, AAT and 3-HPDHactivities as described in herein. The experimental results shown inTable 25.

TABLE 25 Transformant enzyme activity data Gene PYC AAT ADC 3-HPDHStrain Overexpressed activity activity activity activity MBin500 N/A0.14 0.03 0.00 0.14 (control) yMhCt008 ADC (SEQ ID NO: 130), 0.22 2.050.64 2.95 gabT (SEQ ID NO: 141), 3-HPDH (SEQ ID NO: 144) GMEr009-2 ADC(SEQ ID NO: 130), 1.15 24.62 0.05 0.07 PYC (SEQ ID NO: 1), AAT (SEQ IDNO: 13) yMhCt020 ADC (SEQ ID NO: 130), 2.74 35.02 0.56 1.68 GMErin010 #2gabT (SEQ ID NO: 141), 1.97 40.32 0.59 1.87 3-HPDH (SEQ ID NO: 144),GMErin010 #3 PYC (SEQ ID NO: 1), 1.50 23.79 0.42 3.30 AAT (SEQ ID NO:13),

The strains in Table 25 were also analyzed by SDS-PAGE as describedherein. MBin500 and GMEr009-2 showed a protein band a ˜64 kD that wasabsent in the four other samples. The mass of these proteins isconsistent with their identity as the native pyruvate decarboxylase inI. orientalis CNB1. Strains yMhCt008, yMhCt020, GMErin010 #2, andGMErin010 #3 showed bands at 53 kD and 29 kD. The mass of these proteinsis consistent with mass of the proteins encoded by the UGA1 and YMR226cgenes, respectively. Strains GMEr009-2, yMhCt020, GMErin010 #2, andGMErin010 #3 all showed bands at 46.3 kD. The mass of these proteins isconsistent with mass of the protein encoded by the AAT gene.

Example 3A-14: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)from Four Nucleotide Sequences at Both Adh1202 Loci

A ura3-derivative of MeJi409-2 containing four copies of nucleotidesencoding the B. licheniformis ADC of SEQ ID NO: 139 (supra) was isolatedusing the FOA counter-selection loop-out protocol described above.Genomic DNA of several FOA resistant colonies of parent strain MeJi409-2was screened by PCR for the desired loop-out event with primers 0611815and 0611817. Primer 0611815 anneals in the RKI terminator of theleft-hand construct and amplifies toward the ura3 promoter. Primer0611817 anneals in TDH3 promoter and amplifies back toward the ura3cassette. The presence of an 828 bp band indicates the presence of onlythe ura3 scar site (a single URA3 promoter left behind after homologousrecombination between the two URA3 promoters in the parent strain) asdesired, while a band of approximately 2.2 kbp indicates the presence ofthe intact URA3 promoter-URA3 ORF-URA3 terminator-URA3 promotercassette, indicating the desired recombination event did not occur. PCRreactions with Crimson Taq™ DNA polymerase (New England Biolabs) werecarried out as described above. One FOA resistant colony from parentstrain MeJi409-2, designated MeJi411, gave the desired 828 bp band.

Integration of the Left-Hand and Right-Hand Fragments

Plasmid pMeJi310-2 was digested with HpaI and SacII and plasmidpMeJi312-2 was digested with EcoRI and SacII as described herein. Thesewere purified by 1% agarose gel electrophoresis in TBE buffer, and thetwo approximately 5 kbp bands were excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

MeJi411 was transformed with the digested pMeJi310-2 and pMeJi312-2 DNAand correct loci targeting and transformation was verified by CrimsonTaq (New England Biolabs) PCR as described herein. Primers 0611225 and0611632 yielded an approximately 5 kbp band; primers 0611815 and 0611632yielded an approximately 6 kbp band with the ura marker, and a 4.5 kbpband without. Primers 0611631 and 0612579 yield an approximately 936 bpband when the wildtype adh1202 locus is still intact (strains that didnot show this band were selected). A strain which gave the expectedbands for proper integrating of the expression cassette was designatedMeJi412.

Example 3A-15: Yeast Strains Expressing Four Copies of NucleotidesEncoding an Aspartate 1-Decarboxylase (ADC) at the Adh1202 Locus, withTwo Copies of the Nucleotides Encoding the Aspartate 1-Decarboxylase(ADC) Under the Control of the a PDC Promoter and Two Copies Under theControl of a TDH3 Promoter

This example describes constructs designed to incorporate four copies ofnucleotides encoding the B. licheniformis ADC of SEQ ID NO: 139 at theadh1202 locus with two copies of under control of the I. orientalis PDCpromoter and two copies under the control of the I. orientali TDH3promoter. In a similar approach to that described above, a left-hand anda right-hand constructs were designed to allow homologous recombinationat the I. orientalis CNB1 adh1202 locus.

Construction of a Left-Hand Fragment

The plasmid pMeJi310-2 (supra; see FIG. 14) was digested with XbaI andStuI followed by treatment with CIP and purified by 1% agarose gelelectrophoresis in TBE buffer as described herein. A band atapproximately 6.7 kbp was excised from the gel and purified usingQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions.

The PDC promoter was excised from pMeJi310-2 by digestion with NotIfollowed by a fill-in reaction with Klenow and subsequent digestion withNheI. A band at approximately 708 bp was excised from the gel andpurified using QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions.

The 708 bp of purified fragment was ligated into the 6.7 kbp pMeJi310-2linearized vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 1 μL of the 6.7 kbp fragment frompMeJi310-2, 1 or 5 μL of the 708 bp fragment from pMeJi310-2, 1 μL 10×ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4 ligase(New England Biolabs). The reaction was incubated overnight at 16° C.and a 4 μL aliquot of the reaction was transformed into ONE SHOT® TOP10chemically competent E. coli cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated on 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by restriction digestusing ApaLI. A clone yielding correct digested band size was designatedpMIBa137.

Construction of a Right-Hand Fragment

The plasmid pMeJi312-2 (supra) was digested with XbaI and StuI followedby treatment with CIP and purified by 1% agarose gel electrophoresis inTBE buffer as described herein. A band at approximately 6.8 kbp wasexcised from the gel and purified using QIAQUICK® Gel Extraction Kit(Qiagen) according to the manufacturer's instructions.

The TDH3 promoter was excised from pMeJi312-2 by digestion with PmeI andNheI. A band at approximately 966 bp was excised from the gel andpurified using QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions.

The 966 bp of purified fragment was ligated into the 6.8 kbp pMeJi312-2linearized vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 1 μL of the 6.8 kbp fragment frompMeJi312-2, 1 or 5 μL of the 966 bp fragment from pMeJi312-2, 1 μL 10×ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4 ligase(New England Biolabs). The reaction was incubated for approximately 6hours at 16° C. and a 4 μL aliquot of the reaction was transformed intoONE SHOT® TOP10 chemically competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated on2×YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion byrestriction digest using SalI. A clone yielding correct digested bandsize was designated pMIBa136.

Integration of the Left-Hand and Right-Hand Fragments

Plasmid pMIBa137 was digested with HpaI and SacII and plasmid pMIBa136was digested with EcoRI and SacII as described herein. These werepurified by 1% agarose gel electrophoresis in TBE buffer, and the twoapproximately 5 kbp bands were excised from the gel and purified usingQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions.

I. orientalis CNB1 was transformed with the digested pMIBa137 andpMIBa136 DNA and correct loci targeting and transformation was verifiedby Crimson Taq (New England Biolabs) PCR as described in herein. Primers0611717 and 0611631 yielded bands of approximately 2.5 kbp and 955 bp;primers 0611718 and 0611632 yielded an approximately 733 bp band;primers 0612794 and 0611245 yielded an approximately 2.7 kbp band;primers 0611225 and 0612795 yielded an approximately 4.2 kbp band. Astrain which gave the expected bands for proper integration of theexpression cassette was designated MIBa351.

Removal of the Ura Marker from MIBa351

A ura-derivative of MIBa351 was isolated as described above. GenomicDNAs from several FOA resistant colonies of MIBa351 were screened by PCRfor the desired loop-out event with primers 0611815 and 0611817. Primer0611815 anneals in the RKI terminator of the left-hand construct andamplifies toward the ura3 promoter. Primer 0611817 anneals in TDH3promoter and amplifies back toward the ura3 cassette. The presence of an828 bp band indicates the presence of only the ura3 scar site (a singleURA3 promoter left behind after homologous recombination between the twoURA3 promoters in the parent strain) as desired, while a band ofapproximately 2.2 kbp indicates the presence of the intact URA3promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette, indicating thedesired recombination event did not occur. PCR reactions with CrimsonTaq™ DNA polymerase (New England Biolabs) were carried out as describedabove. FOA resistant colonies that yielded the 828 bp fragment with theabove primers were further tested with primers 0612794 and 0611245,which yield a 2.7 kbp product, and primers 0611815 and 0612795, whichyield a 4 kbp product, to confirm that the four copies of the nucleotidesequence SEQ ID NO: 138 encoding the B. licheniformis ADC of SEQ ID NO:139 remained intact. One FOA resistant colony from parent strainMIBa351, designated MIBa353, gave the desired PCR products with all 3primer sets.

Construction of a Reverse Expression Cassette Right-Hand Fragment

Plasmid pMIBa136 contains two expression cassettes going in the forwardorientation.

To ease screening of homozygous strains, a new plasmid was constructedwhere the panDbl expression cassettes of pMIBa136 were placed in thereverse orientation. The plasmid pMIBa136 (supra) was digested with NotIand PmeI followed by a fill-in reaction with Klenow and purified by 1%agarose gel electrophoresis in TBE buffer as described herein. Bands atapproximately 3.4 and 4.4 kbp were excised from the gel and purifiedusing QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions. The 4.4 kbp fragment from pMIBa136 wastreated with CIP and purified using the QIAQUICK® PCR Purification Kit(Qiagen) according to the manufacturer's instructions.

The 3.4 kbp purified fragment from pMIBa136 was ligated into the 4.4 kbppMIBa136 vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 1 μL of the 4.4 kbp fragment frompMIBa136, 1 or 5 μL of the 3.4 kbp fragment from pMIBa136, 1 μL 10×ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4 ligase(New England Biolabs). The reaction was incubated overnight at 16° C.and a 4 μL aliquot of the reaction was transformed into ONE SHOT® TOP10chemically competent E. coli cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated on 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by restriction digestusing PacII and EcoRI. A clone yielding correct digested band size wasdesignated pMIBa138.

Integration of Left-Hand and Right-Hand Fragments

Plasmid pMIBa137 was digested with HpaI and SacII and plasmid pMIBa138was digested with EcoRI and SacII as described herein. These werepurified by 1% agarose gel electrophoresis in TBE buffer, and the twoapproximately 5 kbp bands were excised from the gel and purified usingQIAQUICK® Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions.

MIBa353 was transformed with the digested pMIBa137 and pMIBa138 DNA andcorrect loci targeting and transformation was verified by PCR using thePhire® Plant Direct PCR kit (Finnzymes) according to the manufacturer'sinstructions. Primers 0611718 and 0611632 yielded an approximately 733bp band (to confirm the first integration is still present); primers0612367 and 0611632 yielded an approximately 960 bp band (to confirmthat the second copy integrated); primers 0611631 and 0612579 yielded anapproximately 936 bp band if the wildtype adh1202 locus is still present(lack of this band confirms loss of wt adh1202 locus). Two strains whichgave the expected bands for proper integrating of the expressioncassette were saved and designated MIBa355 and MIBa356.

Aspartate 1-Decarboxylase Activity in MeJi409-2, MeJi412, MIBa351 andMIBa355

Strains MeJi409-2, MeJi412, MIBa351 and MIBa355 were grown in shakeflasks and CFE were prepared and assayed for aspartate 1-decarboxylase(ADC) activity as described herein. The results are shown in Table 26.The activity for each strain is an average of two independent shakeflask cultures. Strains MeJi409-2, MeJi412, MIBa351 and MIBa355 werealso tested in bioreactors for 3-HP production, using the methodsdescribed herein. The results from these bioreactor experiments are alsoshown in Table 26. In order to account for differences in cell mass inthese fermentations, the 3-HP production performance shown in Table 26is expressed as 3-HP concentration per unit of cell mass (expressed asg/L 3-HP/g/L dry cell weight). The results show that as the level of ADCactivity in the cells increased, the 3-HP production performanceincreased.

TABLE 26 Transformant ADC activity and 3-HP production data Strain Geneoverexpressed ADC activity 3-HP/DCW MBin500 NA 0 0 MeJi409-2 ADC (SED IDNO: 138) 0.629 0.19 MeJi412 1.151 0.43 MIBa351 0.659 0.32 MIBa355 1.1730.52

Example 3A-16: Plasmid Construction for Expressing Pyruvate Carboxylase(PYC) at the PDC Locus

Plasmid pANN28 continuing the nucleotide sequence of SEQ ID NO: 1(encoding the I. orientalis PYC of SEQ ID NO: 2) for integration at thePDC locus was constructed as described below.

The upstream and downstream flanking regions of I. orientalis PDC wereamplified by PCR using genomic DNA as a template (Pfu polymerase,Stratagene) according to the manufacturer's instructions. The primersoANN7 and oANN8 allowed the incorporation of unique restriction sitesflanking the upstream region and the primers oANN9 and oANN10 allowedthe incorporation of unique restriction sites flanking the downstreamregion. The PCR products were purified by 1% agarose gel electrophoresisin TBE buffer as described herein. A band of approximately 800 bp foreach PCR product was excised from the gel and purified using a gelextraction kit (Qiagen) according to the manufacturer's instructions.The purified PCR products were cloned into TOPO vectors (Invitrogen) andtransformed into electro-competent E. coli DH10B cells (Invitrogen)according to manufacturer's instructions. Several of the resultingtransformants were screened for proper insertion by colony PCR with thesame primers used to create the PCR products. Positive clones werefurther confirmed by sequencing. A clone yielding the correct PDCdownstream flank was designated pANN04. A clone yielding the correct PDCupstream flank was designated pANN07.

Plasmid pANN04 was digested with ApaI and SacI (for use asvector/backbone); plasmid pANN04 was digested with NotI and SacI;plasmid pANN07 was digested with NotI and ApaI. Each fragment waspurified by 1% agarose gel electrophoresis in TBE buffer as describedherein. A band of approximately 3.5 kbp for the vector, andapproximately 1 kbp for each insert were excised from the gel andpurified using a gel extraction kit (Qiagen) according to themanufacturer's instructions. The purified products were ligated using T4ligase (New England Biolabs) in a total reaction volume of 10 μLcomposed of 49 ng of the vector, 120 ng of the downstream insert, 41 ngof the upstream insert, 1 μL 10× ligation buffer with 10 mM ATP (NewEngland Biolabs), and 1 μL T4 ligase (New England Biolabs). The reactionwas incubated for 30 minutes at room temperature and a 2 μL aliquot ofthe reaction was transformed into electro-competent E. coli OneShotTOP10 cells (Invitrogen) according to manufacturer's instructions.Transformants were plated on LB+Kanamycin plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion by colony PCR with primers oANN7 and oANN10 (yielding aband of approximately 1.7 kbp). A clone yielding the correct insertionwas designated pANN12.

The I. orientalis PYC coding sequence (SEQ ID NO: 1) from pGMEr137(supra) was modified by site directed mutagenesis to eliminate threeEcoRI restriction sites which do not alter the amino acid sequence ofthe encoded enzyme. Plasmid pGMEr137 was used as a template with primersoANN13, oANN14 and oANN15 used to elimination of the above mentionedrestriction sites using a Multi change kit (Stratagene) according to themanufacturer's instructions. Several of the resulting transformants werescreened by restriction digest using EcoRI. Positive clones were furtherconfirmed by sequencing. A clone yielding the correct pyc codingsequence was designated pANN14.

Plasmid pJY39 (FIG. 29) was digested with XhoI and PacI; plasmid pACN5(supra; see FIG. 19) was digested with XhoI and XbaI; plasmid pANN14 wasdigested with XbaI and PacI. Each fragment was purified by 1% agarosegel electrophoresis in TBE buffer as described herein. A band ofapproximately 8 kbp for the vector, approximately 700 bp for the firstinsert, and approximately 3.6 kbp for the second insert encoding the PYCwere excised from the gel and purified using a gel extraction kit(Qiagen) according to the manufacturer's instructions. The purifiedproducts were ligated using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 51 ng of the vector, 49 ng of thefirst insert, 210 ng of the second insert, 1 μL 10× ligation buffer with10 mM ATP (New England Biolabs), and 1 μL T4 ligase (New EnglandBiolabs). The reaction was incubated for 30 minutes at room temperatureand a 2 μL aliquot of the reaction was transformed intoelectro-competent E. coli OneShot TOP10 cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated on LB+Kanamycinplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by colony PCR withprimers oJLJ57 and oJLJ43 (yielding a band of approximately 1 kbp),primers oJLJ45 and oANN16 (yielding a band of approximately 730 bp), andprimers oANN20 and oJY45 (yielding a band of approximately 1.2 kbp). Aclone yielding the correct insertion was designated pANN15.

Plasmids pANN12 and pANN15 were digested with NotI. Plasmid pANN15 wasadditionally digested with NcoI for further fractionation of thebackbone and improved separation of desired fragment. The digestedpANN12 was purified using a Qiagen kit according to the manufacturer'sinstructions. The NotI fragments were purified by agarose gelelectrophoresis in TBE buffer as described herein. The bands ofapproximately 5 kbp (from pANN12) and approximately 6.3 kbp (frompANN15) were gel purified using a gel extraction kit (Qiagen) accordingto the manufacturer's instructions.

The purified product from pANN15 was ligated into the pANN12 linearizedvector using T4 ligase (New England Biolabs) in a total reaction volumeof 10 μL composed of 50 ng of the vector, 115 ng of the insert, 1 μL 10×ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4 ligase(New England Biolabs). The reaction was incubated for 1.5 hr at roomtemperature and a 2 μL aliquot of the reaction was transformed intoelectro-competent E. coli OneShot TOP10 cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated on LB+Kanamycinplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by colony PCR withprimers oANN20 and oJY45 (yielding a band of approximately 1.2 kbp).Clones yielding correct insertion were further screened by restrictionenzyme digestion with SacI/EcoRI and with SacI/EcoRV in order todifferentiate the insert orientation. A clone yielding the ura3 markernear the upstream PDC flank was designated pANN27. A clone yielding theura3 marker near the downstream PDC flank was designated pANN28.

Example 3A-17: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)from Four Nucleotide Sequences at the Adh1202 Locus and PyruvateCarboxylase (PYC) at the Pdc Locus

This example describes the construction of yeast strains expressing fourcopies of nucleotides encoding the B. licheniformis ADC of SEQ ID NO:139 at the adh1202 locus and a nucleotide encoding the I. orientalis PYCof SEQ ID NO: 2 at the pdc locus.

Removal of the Ura Marker from MeJi412

A ura-derivative of MeJi412 was isolated as described above. Several FOAresistant colonies of MeJi412 were screened by colony PCR for thedesired loop-out event with primers 0611815 and 0611817. Primer 0611815anneals in the RKI terminator of the left-hand construct and amplifiestoward the ura3 promoter. Primer 0611817 anneals in TDH3 promoter andamplifies back toward the ura3 cassette. The presence of an 869 bp bandindicates the presence of only the ura3 scar site (a single URA3promoter left behind after homologous recombination between the two URA3promoters in the parent strain) as desired, while a band ofapproximately 2.6 kbp indicates the presence of the intact URA3promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette, indicating thedesired recombination event did not occur. PCR reactions with Phire®Plant Direct PCR Kit (Finnzymes) were carried out as described above.FOA resistant colonies that yielded the 869 bp fragment with the aboveprimers were further tested with primers 0612794 and 0611817, whichyield a 3.8 kbp product, and primers 611815 and 612795, which yield a3.7 kbp product, to confirm that the four copies of the nucleotidesequence SEQ ID NO: 138 encoding the B. licheniformis ADC of SEQ ID NO:139 remained intact. One FOA resistant colony from parent strainMeJi412, designated MeJi413, gave the desired PCR products with all 3primer sets.

Integration of Fragment

Plasmid pANN28 (supra) was digested with AscI and SacI and purified byagarose gel electrophoresis in TBE buffer. The band at approximately 7.1kbp was excised from the gel and purified using a gel purification kit(Qiagen) according to the manufacturer's instructions.

Strain MeJi413 was transformed with the digested and purified fragmentfrom pANN28 and correct loci targeting and transformation was verifiedby colony PCR (Failsafe, mix E, Epicenter) according to themanufacturer's instructions. Primers oANN12 and oJLJ44 yielded anapproximately 1 kbp band; primers oANN11 and oANN16 yielded anapproximately 1.3 kbp band. A strain which gave the expected bands forproper integration of the expression cassette was designated yANN35.

A ura-derivative of yANN35 then was isolated as described above. SeveralFOA resistant colonies were screened by colony PCR for the desiredloop-out event with primers oANN12 and oJY44. Primer oANN12 annealsoutside of the downstream flanking region. Primer oJY44 anneals to theTAL terminator. The presence of a 1.5 kbp band indicates the presence ofonly the ura3 scar site (a single URA3 promoter left behind afterhomologous recombination between the two URA3 promoters in the parentstrain) as desired, while a band of approximately 2.8 kbp indicates thepresence of the intact URA3 promoter-URA3 ORF-URA3 terminator-URA3promoter cassette, indicating the desired recombination event did notoccur. PCR reactions with Failsafe DNA polymerase (Epicenter) werecarried out as described above. Isolates positive for this event werefurther confirmed by colony PCR with primers oANN16 and oANN11. One FOAresistant colony was designated yANN37.

Strain yANN37 was transformed with the digested and purified fragmentsfrom pANN28 and correct loci targeting and transformation was verifiedby colony PCR (Failsafe, mix E, Epicenter) according to themanufacturer's instructions. The preliminary screen was done withprimers oHJJ116 and oHJJ117 which are specific for the PDC gene. A bandof approximately 500 bp indicates the presence of the gene and thus anegative result for the desired integration. Isolates that were positivefor deletion of PDC were further confirmed with additional PCRreactions. Primers oANN11 and oANN16 yielded an approximately 1.3 kbpband. Primers oANN12 and oJLJ44 yielded an approximately 1 kbp band;primers oANN12 and oJY44 yielded an approximately 1.5 kbp band and anapproximately 2.9 kbp band (corresponding to the first and secondintegration events respectively).

Additionally, the previous integration events at the adh1202 locus wereconfirmed by colony PCR as described above. Primers 0611631 and 0611245yielded an approximately 3.8 kbp band. Primers 0611245 and oNovo3yielded an approximately 3 kbp band. Primers 0611815 and 0612795 yieldedan approximately 3.6 kbp band. A strain which gave the expected bandsfor proper integration of the expression cassette was designated yANN41.

Example 3A-18: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)from Four Nucleotide Sequences at the Adh1202 Locus and PyruvateCarboxylase (PYC) at the Pdc Locus

This example describes the construction of yeast strains expressing fourcopies of nucleotides encoding the B. licheniformis ADC of SEQ ID NO:139 at the adh1202 locus (with two copies under the control of the a PDCpromoter and two copies under the control of a TDH3 promoter) and anucleotide encoding the I. orientalis PYC of SEQ ID NO: 2 at the pdclocus.

Removal of Ura Marker from MIBa355

A ura-derivative of MIBa355 was isolated as described above. Genomic DNAfrom several FOA resistant colonies of MIBa355 were screened by PCR forthe desired loop-out event with primers 0611815 and 0611718. Thepresence of an approximately 500 bp band indicates the presence of onlythe ura3 scar site (a single URA3 promoter left behind after homologousrecombination between the two URA3 promoters in the parent strain) asdesired, while a band of approximately 1.9 kbp indicated the presence ofthe intact URA3 promoter-URA3 ORF-URA3 terminator-URA3 promotercassette, indicating the desired recombination event did not occur. PCRwas performed using the Phire® Plant Direct PCR kit (Finnzymes)according to the manufacturer's instructions. FOA resistant coloniesthat yielded the approximately 500 bp fragment with the above primerswere further tested with primers 0611631 and 0611245, which yield a 3.5kbp product, and primers 0611815 and 0611632, which yield a 4.5 kbpproduct, to confirm that the four copies of the nucleotide sequence SEQID NO: 138 encoding the B. licheniformis ADC of SEQ ID NO: 139 remainedintact. They were also tested with PCR using primers 0611815 and 0611817to confirm that the first modification at adh1202 was present. These PCRprimers yielded a 828 bp fragment. One FOA resistant colony from parentstrain MIBa355, designated MIBa357, gave the desired PCR products withall four primer sets.

The plasmid pANN28 (supra) was digested with AscI and SacI and purifiedby 1% agarose gel electrophoresis in TBE buffer. Approximately 7.1 kbpband was excised from the gel and purified using a NUCLEOSPIN® ExtractII Kit (Macherey-Nagel) according to the manufacturer's instructions.

MIBa357 was transformed with the digested pANN28 DNA and correct locitargeting and transformation was verified by PCR using Phire PlantDirect PCR Kit (Finnzymes). Primers 0611622 and 0611552 yielded anapproximately 850 bp band; primers 0611245 and 0612794 yielded anapproximately 2.8 kbp band; primers 0611815 and 0612795 yielded anapproximately 3.9 kbp band. A strain which gave the expected bands forproper integrating of the expression cassette was designated McTs241.

A ura-derivative of McTs241 then was isolated as described previously.Several FOA resistant colonies of McTs241 were screened by PCR for thedesired loop-out event with primers 0614233 and 0611554 and lack ofgrowth on ura minus selection plates. The presence of an 4.6 kbp bandindicated the presence of only the ura3 scar site (a single URA3promoter left behind after homologous recombination between the two URA3promoters in the parent strain) as desired, while a band ofapproximately 5.9 kbp indicated the presence of the intact URA3promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette, indicating thedesired recombination event did not occur. PCR reactions using PhirePlant Direct PCR Kit (Finnzymes) were carried out as described above.One FOA resistant colony from parent strain McTs241 that had the desiredloop-out event was designated McTs247.

To create the homozygous integration McTs247 was transformed with thedigested pANN28 DNA and correct loci targeting and transformation wasverified by PCR using Phire Plant Direct PCR Kit (Finnzymes). As a firstscreen transformants were screen by PCR with primers 0611552 and 0611553which should yield an approximately 850 bp band only if the pdc locus isintact and thus the homozygous integration of PYC at the PDC locus didnot occur. Of those that were negative for a band from this PCR werethen screened by additional PCR with primers 0611555 and 0611554. Withthese primers a product should only amplify 1.4 kbp band if PDC isintact and thus not a homozygous integration of PYC at PDC locus.Further screening of transformants was done by PCR using primers 0611622and 0611552 yielding an approximately 850 bp band; primers 0611245 and0612794 yielded an approximately 2.8 kbp band; primers 0611815 and0612795 yielded an approximately 3.9 kbp band. A strain which gave theexpected bands for proper integrating of the expression cassette wasdesignated McTs253.

Example 3A-19: PYC Activity, ADC Activity and 3-HP ProductionPerformance of Strains MeJi412, yANN35, yANN41, MIBa355, McTs241 andMcTs253

Strains MeJi412, yANN35, yANN41, MIBa355, McTs241 and McTs253 were grownin shake flasks and CFE were prepared and assayed for pyruvatecarboxylase (PYC) activity and aspartate 1-decarboxylase (ADC) activityas described herein. The results are shown in Table 27. Strains MeJi412,yANN35, yANN41, MIBa355, McTs241 and McTs253 were also tested inbioreactors for 3-HP production, using the methods described herein. Theresults from these bioreactor experiments are also shown in Table 27. Inorder to account for differences in cell mass in these fermentations,the 3-HP production performance shown is expressed as 3-HP concentrationper unit of cell mass (expressed as g/L 3-HP/g/L dry cell weight). Theresults show that as the level of PYC activity in the cells increased,the 3-HP production performance increased.

TABLE 27 Transformant PYC and ADC activity and 3-HP productionperformance PYC ADC Strain Gene Overexpressed activity activity 3-HP/DCWMeJi412 ADC (SED ID NO: 138) 6.8 1.151 0.43 yANN35 ADC (SED ID NO: 138)47.2 1.090 0.64 PYC (SEQ ID NO: 1) yANN41 ADC (SED ID NO: 138) 49.01.263 1.30 PYC (SEQ ID NO: 1) MIBa355 ADC (SED ID NO: 138) 6.9 1.1730.52 McTs241 ADC (SED ID NO: 138) 24.8 1.119 0.76 PYC (SEQ ID NO: 1)McTs253 ADC (SEQ ID NO: 138) 55.5 1.347 1.30 PYC (SEQ ID NO: 1)

Example 3A-20: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)from Four Nucleotide Sequences at the Adh1202 Locus and Deletion ofBeta-Alanine Aminotransferase (BAAT)

This example describes the construction and performance of yeast strainsexpressing four copies of nucleotides encoding the B. licheniformis ADCof SEQ ID NO: 139 at the adh1202 locus and deletion of the native I.orientalis gene encoding the BAAT (PYD4) of SEQ ID NO: 20.

Construction of I. Orientalis BAAT (PYD4) Deletion Plasmid

The plasmid pMIBa123 (supra) was digested with NotI, KpnI, ApaI and thepurified by 1% agarose gel electrophoresis in TBE buffer as describedherein. Two bands at approximately 3.6 kbp and 3.8 kbp were excised fromthe gel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions. These two pieces comprisedthe plasmid backbone and the ura selection cassette with S. cerevisiae3-HPDH gene (YMR226c) of SEQ ID NO: 144.

A PCR product for the upstream I. orientalis PYD4 homology was generatedby PCR amplification using I. orientalis MBin500 genomic DNA prepared asdescribed previously using primers 0613178 and 0613180. The downstreamI. orientalis PYD4 homology piece was prepared by PCR amplification fromMBin500 genomic DNA using primers 0613179 and 0613181. Fifty μmoles ofeach primer was used in a PCR reaction containing 0.5 μL of MBin500genomic DNA as template, 0.2 mM each dATP, dGTP, dCTP, dTTP, 1× ExpandHigh Fidelity Buffer (Roche), 3.5 U Expand High Fidelity Enzyme Mix(Roche) in a final volume of 50 μL. The amplification reaction wasperformed in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf) programmed forone cycle at 95° C. for 3 minutes; and 30 cycles each at 95° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. After thecycles, the reaction was incubated at 72° C. for 5 minutes and thencooled at 10° C. until further processed. The approximately 800 bp bandfrom PCR using primers 0613178 and 0613180 and the approximately 900 bpband from PCR using primers 0613179 and 0613181 were purified by 1%agarose gel electrophoresis in TBE buffer. The bands were excised fromthe gel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions.

The PYD4 upstream PCR product, PYD4 downstream PCR product, and pMIBa123NotI/KpnI/ApaI digested plasmid were assembled in a reaction withIN-FUSION HD™ (Clontech Laboratories, Inc.) according to manufacturer'sinstructions. From the In-FUSION reaction 2 μL was transformed into ONESHOT® TOP10 chemically competent E. coli cells (Invitrogen) according tomanufacturer's instructions. After a recovery period, two 100 μLaliquots from the transformation reaction were plated onto 150 mm 2× YTplates supplemented with 100 μg of ampicillin per mL. The plates wereincubated overnight at 37° C. Putative recombinant clones were selectedfrom the selection plates and plasmid DNA was prepared from each oneusing a BIOROBOT® 9600 (Qiagen). Clones were analyzed by restrictiondigest and sequencing. A plasmid with the correct sequence were verifiedby sequencing and named pMcTs61.

The plasmid pMcTs61 still contains the PDC promoter, YMR226c gene fromS. cerevisiae, and the PDC terminator. To remove these undesiredsegments, pMcTs61 was digested with EcoRI and XhoI followed by additionof Klenow fragment to create blunt ends. The 7.1 kbp fragment waspurified by 1% agarose gel electrophoresis in TBE buffer. The band wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions. Thedigested blunt plasmid was ligated together using T4 DNA ligase (NewEngland Biolabs, Ipswich, Mass., USA). The reaction mixture contained 1×T4 DNA ligase buffer, 1 μL T4 DNA ligase, 5 μL pMcTs61 digested andblunted purified DNA in total volume of 20 μL. The reaction wasincubated at room temperature for 2 hours. A 10 μL sample of theligation reaction was used to transform ONE SHOT® TOP10 chemicallycompetent E. coli cells (Invitrogen) according to according to themanufacturer's instructions. After a recovery period, two 100 μLaliquots from the transformation reaction were plated onto 150 mm 2× YTplates supplemented with 100 μg of ampicillin per mL. The plates wereincubated overnight at 37° C. Putative recombinant clones were selectedfrom the selection plates. Clones were analyzed by colony PCR. TemplateDNA from each colony was prepared by dissolving 1 colony in 50 μLsterile water, heated at 95° C. for 10 minutes, then cooled on ice untiluse. Primers 0612911 and 0612909 were used to screen the transformants.The PCR reaction with these primers would amplify a 1 kbp band if theplasmid was correct. Ten μmoles of each primer was used in a PCRreaction containing 2 μL colony DNA template, 0.1 mM each dATP, dGTP,dCTP, dTTP, 1× Crimson Taq Reaction Buffer (New England Biolabs), 1 UCrimson Taq DNA Polymerase (New England Biolabs) in a final volume of 20μL. The amplification reaction was performed in an EPPENDORF®MASTERCYCLER® 5333 (Eppendorf) programmed for one cycle at 95° C. for 3minutes; and 30 cycles each at 95° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 3 minutes. After the cycles, the reaction wasincubated at 72° C. for 5 minutes and then cooled at 10° C. untilfurther processed. From 5 μL of the PCR reaction a 1 kbp PCR fragmentwas visualized on a 1% TAE-agarose gel with ethidium bromide in TAEbuffer. One transformant with the correct size PCR product was selectedand named pMcTs64 (FIG. 30). Plasmid DNA of pMcTs64 was prepared using aBIOROBOT® 9600 (Qiagen).

Deletion of Native I. orientalis BAAT (PYD4) from MeJi413 Using pMcTs64Construct

Plasmid pMcTs64 (supra; see FIG. 30) was digested with ApaI, NcoI, KpnIand purified by 1% agarose gel electrophoresis in TBE buffer.Approximately 3.3 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

Strain MeJi413 (supra) was transformed with the digested pMcTs64 DNA andcorrect loci targeting and transformation was verified by PCR usingPhire Plant Direct PCR Kit (Finnzymes). Primers 0612908 and 0613242yield an approximately 1.7 kbp band; primers 0613241 and 0612909 yieldan approximately 1.5 kbp band to confirm the integration of the deletioncassette. Primers 0611815 and 0611632 yield an approximately 4.2 kbpband; primers 0611817 and 0611631 yield an approximately 4.8 kbp band toconfirm the ADC cassette at the ADH1202 locus was still intact. A strainwhich gave the expected bands for proper integrating of the deletioncassette and ADC cassette was designated McTs225.

A ura-derivative of McTs225 then was isolated as described previously.Several FOA resistant colonies of McTs225 were screened by PCR for thedesired loop-out event with primers 0612911 and 0612910. The presence ofan 1.1 kbp band indicates the presence of only the ura3 scar site (asingle URA3 promoter left behind after homologous recombination betweenthe two URA3 promoters in the parent strain) as desired, while a band ofapproximately 2.5 kbp indicates the presence of the intact URA3promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette, indicating thedesired recombination event did not occur. Primers 0611815 and 0611632yield an approximately 4.2 kbp band; primers 0611817 and 0611631 yieldan approximately 4.8 kbp band to confirm the ADC cassette at the adh1202locus was still intact. PCR reactions using Phire Plant Direct PCR Kit(Finnzymes) were carried out as described above. One FOA resistantcolony from parent strain McTs225 that had the desired loop-out eventwas designated McTs228.

To create a homozygous deletion of the native gene encoding the I.orientalis BAAT (PYD4) of SEQ ID NO: 20, McTs228 was transformed withdigested pMcTs64 and correct loci targeting and transformation wasverified by PCR using Phire Plant Direct PCR Kit (Finnzymes). Two primersets were used to screen by PCR for PYD4 locus deletion. Primers 0613550and 0612910 yield an approximately 700 bp band only if the PYD4 locus isintact which would indicate that homozygous deletion of PYD4 did notoccur. Additionally transformants were screen with primers 0612911 and0613551 which yield an approximately 600 bp band if PYD4 was notdeleted. Transformants that were negative for the I. orientalis PYD4locus were further screened with primers 0613242 and 0613243 yielding anapproximately 3.5 kbp and 2.1 kbp band; primers 0612908 and 0613243yielded an approximately 1.7 kbp band; primers 0612909 and 0612911yielded an approximately 950 bp band. The ADC cassette at adh1202 locuswas confirmed to still be intact with primers 0611817 and 0611631yielding an approximate 4.8 kbp and primers 611815 and 612712 yieldingan approximate 4.2 kbp band. A strain which gave the expected bands forproper integrating of the expression cassette was designated McTs236.

A ura-derivative of McTs236 then was isolated as described previously.Several FOA resistant colonies of McTs236 were screened by PCR for thedesired loop-out event with primers 0613242 and 0613243 yielding anapproximately 2.1 kbp band. The ADC cassette at ADH1202 locus wasconfirmed to still be intact with primers 0611245 and 0612794 yieldingan approximate 3 kbp and primers 0611815 and 0612795 yielding anapproximate 3.6 kbp band. A strain which gave the expected bands forproper integrating of the expression cassette was designated McTs245.

Strains MIBa372 and McTs245 were tested in bioreactors for 3-HPproduction, using the methods described herein. In order to account fordifferences in cell mass in these fermentations, the 3-HP productionperformance is expressed as 3-HP concentration per unit of cell mass(expressed as g/L 3-HP/g/L dry cell weight). The g/L 3-HP/g/L dry cellweight for strains MIBa372 and McTs245 were 1.66 and 0.16, respectively.These results suggest that the native PYD4 gene in I. orientalis isresponsible for the conversion of beta-alanine to malonate semialdehyde,since deletion of this gene led to a 10-fold decrease in 3-HP productionperformance.

Example 3A-21: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC)from Four Nucleotide Sequences at the Adh1202 Locus and Deletion of 3-HPDehydrogenase (3-HPDH)

This example describes the construction and performance of yeast strainsexpressing four copies of nucleotides encoding the B. licheniformis ADCof SEQ ID NO: 139 at the adh1202 locus and deletion of the native I.orientalis gene encoding the 3-HPDH of SEQ ID NO: 26.

Construction of I. orientalis 3-HPDH Deletion Plasmid

The plasmid pMIBa123 (supra) was digested with NotI, KpnI, ApaI and thepurified by 1% agarose gel electrophoresis in TBE buffer as describedherein. Two bands at approximately 3.6 kbp and 3.8 kbp were excised fromthe gel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions. These two pieces comprisedthe plasmid backbone and the ura selection cassette with S. cerevisiae3-HPDH gene of SEQ ID NO: 144.

A PCR product for the upstream I. orientalis 3-HPDH homology wasamplified from I. orientalis MBin500 genomic DNA prepared as describedpreviously using primers 0613183 and 0613184. A downstream I. orientalis3-HPDH homology piece was amplified from I. orientalis MBin500 genomicDNA using primers 0613185 and 0613186. Fifty pmol of each primer wasused in a PCR reaction containing 0.5 μl of MBin500 genomic DNA astemplate, 0.2 mM each dATP, dGTP, dCTP, dTTP, 2% DMSO, 1× Phusion HFBuffer (FinnzymeS), 2U Phusion® Hot Start High-Fidelity DNA Polymerase(Finnzymes) in a final volume of 50 μl. The amplification reaction wasperformed in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific,Inc., Westbury, N.Y., USA) programmed for one cycle at 95° C. for 3minutes; and 30 cycles each at 95° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute. After the cycles, the reaction wasincubated at 72° C. for 5 minutes and then cooled at 4° C. until furtherprocessed. The approximately 640 bp band from PCR of primers 0613183 and0613184 and the approximately 670 bp band of the PCR from primers0613185 and 0613186 was purified by 1% agarose gel electrophoresis inTBE buffer. The bands were excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The I. orientalis 3-HPDH upstream PCR product, I. orientalis 3-HPDHdownstream PCR product, and pMIBa123 NotI/KpnI/ApaI digested plasmidwere assembled in a reaction with IN-FUSION HD™ (Clontech Laboratories,Inc.) according to manufacturer's instructions. From the In-FUSIONreaction 2 μL was transformed into ONE SHOT® TOP10 chemically competentE. coli cells (Invitrogen) according to manufacturer's instructions.After a recovery period, two 100 μl aliquots from the transformationreaction were plated onto 150 mm 2× YT plates supplemented with 100 μgof ampicillin per mL. The plates were incubated overnight at 37° C.Putative recombinant clones were selected from the selection plates andplasmid DNA was prepared from each one using a BIOROBOT® 9600 (Qiagen).Clones were analyzed by restriction digest and sequencing. A plasmidwith the correct sequence was verified by sequencing and named pMcTs60.

The plasmid pMcTs60 still contains the PDC promoter, YMR226c gene fromS. cerevisiae, and the PDC terminator. To remove these undesiredsegments, pMcTs60 was digested with NotI and XbaI and the approximately5 kbp band containing the 3-HPDH homology regions and the plasmidbackbone were purified by 1% agarose gel electrophoresis in TBE buffer.The bands were excised from the gel and purified using a NUCLEOSPIN®Extract II Kit (Macherey-Nagel) according to the manufacturer'sinstructions. The ura3 selection cassette was amplified with 2 PCRreactions one with primers 0613416 and 0613417 and the other withprimers 0613418 and 0613419. Fifty pmol of each primer was used in a PCRreaction containing 0.5 μL of pMcTs60 plasmid DNA as template, 0.2 mMeach dATP, dGTP, dCTP, dTTP, 1× Expand High Fidelity Buffer (Roche), 3.5U Expand High Fidelity Enzyme Mix (Roche) in a final volume of 50 μL.The amplification reaction was performed in an EPPENDORF® MASTERCYCLER®5333 (Eppendorf Scientific, Inc.) programmed for one cycle at 95° C. for3 minutes; and 30 cycles each at 95° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 1 minute. After the cycles, the reaction wasincubated at 72° C. for 5 minutes and then cooled at 4° C. until furtherprocessed. The approximately 700 bp band from PCR of primers 0613416 and0613417 and the approximately 1 kbp band of the PCR from primers 0613418and 0613419 was purified by 1% agarose gel electrophoresis in TBEbuffer. The bands were excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions. The plasmid backbone containing the I.orientalis 3-HPDH homology regions and the ura3 cassette PCR productswere assembled in a reaction with IN-FUSION HD™ (Clontech Laboratories,Inc.) according to manufacturer's instructions. From the In-FUSIONreaction 2 μL was transformed into Solo Pack Gold Super Competent Cells(Stratagene) according to manufacturer's instructions. After a recoveryperiod, two 100 μL aliquots from the transformation reaction were platedonto 150 mm 2× YT plates supplemented with 100 μg of ampicillin per mL.The plates were incubated overnight at 37° C. Putative recombinantclones were selected from the selection plates and plasmid DNA wasprepared from each one using a BIOROBOT® 9600 (Qiagen). Clones wereanalyzed by restriction digest and a plasmid with the correctrestriction digest pattern was named pMcTs65 (FIG. 31).

Deletion of Native I. orientalis 3-HPDH from MeJi413 Using pMcTs65Construct

Plasmid pMcTs65 (supra; see FIG. 31) was digested with ApaI, Sph, KpnIand purified by 1% agarose gel electrophoresis in TBE buffer.Approximately 2.9 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

Strain MeJi413 (supra) was transformed with the digested pMcTs65 DNA andcorrect loci targeting and transformation was verified by PCR usingPhire Plant Direct PCR Kit (Finnzymes). Primers 0613034 and 0613035yielded an approximately 2.7 kbp band to confirm the integration of thedeletion cassette. Primers 0611815 and 0611632 yielded an approximately4.2 kbp band; primers 0611817 and 0611631 yielded an approximately 4.8kbp band to confirm the ADC cassette at the adh1202 locus was stillintact. A strain which gave the expected bands for proper integrating ofthe deletion cassette and ADC cassette was designated McTs229.

A ura-derivative of McTs229 then was isolated as described previously.Several FOA resistant colonies of McTs229 were screened by PCR for thedesired loop-out event with primers 0613034 and 0613241. The presence ofan 1.4 kbp band indicates the presence of only the ura3 scar site (asingle URA3 promoter left behind after homologous recombination betweenthe two URA3 promoters in the parent strain) as desired, while a band ofapproximately 2.8 kbp indicates the presence of the intact URA3promoter-URA3 ORF-URA3 terminator-URA3 promoter cassette, indicating thedesired recombination event did not occur. The presence of a 1.9 kbpband indicates the wild-type locus which is present in thesetransformants since these are heterozygous for the deletion. Primers0611815 and 0611632 yielded an approximately 4.2 kbp band; primers0611631 and 0612366 yielded an approximately 4.5 kbp band to confirm theADC cassette at the ADH1202 locus was still intact. PCR reactions usingPhire Plant Direct PCR Kit (Finnzymes) were carried out as describedabove. One FOA resistant colony from parent strain McTs225 that had thedesired loop-out event was designated McTs238.

To create a homozygous deletion of the native gene encoding the I.orientalis 3-HPDH of SEQ ID NO: 26, McTs238 was transformed withdigested pMcTs65 and correct loci targeting and transformation wasverified by PCR using Phire Plant Direct PCR Kit (Finnzymes). Two primersets were used to screen by PCR for YMR226c locus deletion. Primers0613034 and 0613747 would yield an approximately 500 bp band if the3-HPDH locus is intact to indicate that homozygous deletion of 3-HPDHdid not occur. Additionally transformants were screened with primers0613746 and 0613241 which would yield an approximately 660 bp band if3-HPDH was not deleted. Transformants that were wild-type negative forthe I. orientalis 3-HPDH locus were further screened with primers0613034 and 0613241 yielding an approximately 2.8 kbp and 1.4 kbp band;primers 0612908 and 0613241 yielded an approximately 1.5 kbp band;primers 0613034 and 0612909 yielded an approximately 1 kbp band. The ADCcassette at ADH1202 locus was confirmed to still be intact with primers0611245 and 0612794 yielding an approximate 3 kbp and primers 0611815and 0612795 yielding an approximate 3.6 kbp band. A strain which had theexpected bands for proper integrating of the expression cassette wasdesignated McTs244.

A ura-derivative of McTs244 when as isolated as described previously.Several FOA resistant colonies of McTs244 were screened by PCR for thedesired loop-out event with primers 0613034 and 0613241 yielding anapproximately 1.4 kbp band. The ADC cassette at ADH1202 locus wasconfirmed to still be intact with primers 0611245 and 0612794 yieldingan approximately 3 kbp band and primers 0611815 and 0612795 yielding anapproximately 3.6 kbp band. One FOA resistant colony from parent strainMcTs244 that had the desired loop-out event was designated McTs259.

Strains MIBa372 and McTs244 were tested in bioreactors for 3-HPproduction, using the methods described herein. In order to account fordifferences in cell mass in these fermentations, the 3-HP productionperformance is expressed as 3-HP concentration per unit of cell mass(expressed as g/L 3-HP/g/L dry cell weight). The g/L 3-HP/g/L dry cellweight for strains MIBa372 and McTs259 were 1.66 and <0.1, respectively.These results indicate that the native 3-HPDH gene in I. orientalis isresponsible for the conversion of malonate semialdehyde to 3-HP, sincedeletion of this gene abolished 3-HP production.

Example 3A-22: Yeast Strains Expressing Pyruvate Carboxylase (PYC),Aspartate Aminotransferase (AAT), β-Alanine Aminotransferase (BAAT), and3-Hydroxypropionic Acid Dehydrogenase (3-HPDH) at the Pdc Locus; andAspartate 1-Decarboxylase (ADC) from Four Nucleotide Sequences at theAdh1202 Locus

Additional constructs were designed to incorporate the nucleotidesequence SEQ ID NO: 1 encoding the I. orientalis PYC of SEQ ID NO: 2,the nucleotide sequence SEQ ID NO: 13 encoding the I. orientalis AAT ofSEQ ID NO: 14, the nucleotide sequence SEQ ID NO: 142 encoding the S.kluyveri BAAT of SEQ ID NO: 21, and the nucleotide sequence SEQ ID NO:144 encoding the S. cerevisiae 3-HPDH of SEQ ID NO: 129 at the I.orientalis pdc locus in stains that also contain four copies ofnucleotides encoding the B. licheniformis ADC of SEQ ID NO: 139 at theadh1202 locus. In a similar approach to that described above, aleft-hand and a right-hand construct were designed to allow homologousrecombination at the I. orientalis CNB1 pdc locus. These constructs wereprepared and transformed into MIBa357 as described below.

Construction of a Left-Hand Fragment

The nucleotide sequence SEQ ID NO: 13 encoding the I. orientalis AAT ofSEQ ID NO: 14 was amplified by PCR using plasmid pGMEr126 (FIG. 16) as atemplate according to the manufacturer's instructions (Pfu polymerase,Stratagene). The primers oANN1 and oANN2 allowed the incorporation ofunique restriction sites flanking the gene coding sequence. The PCRproduct was purified by 1% agarose gel electrophoresis in TBE buffer asdescribed herein. A band of approximately 1.3 kbp was excised from thegel and purified using a gel extraction kit (Qiagen) according to themanufacturer's instructions. The purified PCR product was digested withApaI and NruI and gel purified as described herein.

The plasmid pGMEr135 (identical to pGMEr136 above, except that the ENO1promoter/RKI terminator insert is in opposite orientation) was digestedwith ApaI and NruI and purified by agarose gel electrophoresis in TBEbuffer as described herein. A band of approximately 11.7 kbp was excisedfrom the gel and purified using a gel extraction kit (Qiagen) accordingto the manufacturer's instructions.

The purified 1.3 kbp PCR product was ligated into the 11.7 kbp pGMEr135linearized vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 49.8 ng of the vector, 354 ng ofthe 1.3 kbp insert, 1 μL 10× ligation buffer with 10 mM ATP (New EnglandBiolabs), and 1 μL T4 ligase (New England Biolabs). The reaction wasincubated for 30 minutes at room temperature and a 2 μL aliquot of thereaction was transformed into electro-competent E. coli DH10B cells(Invitrogen) according to manufacturer's instructions. Transformantswere plated on LB+Kanamycin plates and incubated at 37° C. overnight.Several of the resulting transformants were screened for properinsertion by colony PCR with primers oHJ2 and oANN1 (yielding a band ofapproximately 2.3 kbp) and primers oANN5 and oANN6 (yielding a band ofapproximately 877 bp). The sequence of the aat fragment amplified by PCRwas also confirmed. A clone yielding correct insertion and sequence wasdigested with ApaI and NruI and gel purified as described herein. A bandof approximately 1.3 kbp was excised from the gel and purified using agel extraction kit (Qiagen) according to the manufacturer'sinstructions.

The plasmid pGMEr137 (supra; see FIG. 18), containing the desirednucleotide sequence SEQ ID NO: 1 encoding the I. orientalis PYC of SEQID NO: 2, was digested with ApaI and NruI and purified by agarose gelelectrophoresis in TBE buffer as described herein. A band ofapproximately 11.7 kbp was excised from the gel and purified using a gelextraction kit (Qiagen) according to the manufacturer's instructions.

The purified 1.3 kbp PCR product was ligated into the 11.7 kbp pGMEr137linearized vector using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 49.4 ng of the vector, 54 ng of the1.3 kbp insert, 1 μL 10× ligation buffer with 10 mM ATP (New EnglandBiolabs), and 1 μL T4 ligase (New England Biolabs). The reaction wasincubated for 30 minutes at room temperature and a 2 μL aliquot of thereaction was transformed into electro-competent E. coli DH10B cells(Invitrogen) according to manufacturer's instructions. Transformantswere plated on LB+Kanamycin plates and incubated at 37° C. overnight.Several of the resulting transformants were screened for properinsertion by colony PCR with primers oHJ2 and oANN1 (yielding a band ofapproximately 2.3 kbp) and primers oANN5 and oANN6 (yielding a band ofapproximately 877 bp). A clone yielding correct insertion and sequencewas designated pANN02.

Construction of a Left-Hand Fragment with the AAT Encoding Sequence inthe Opposite Orientation

The plasmid pANN02 was digested with PmeI and purified by agarose gelelectrophoresis in TBE buffer as described herein. A band ofapproximately 10.3 kbp and a band of approximately 2.7 kbp were excisedfrom the gel and purified using a gel extraction kit (Qiagen) accordingto the manufacturer's instructions. The 10.3 kbp vector fragment frompANN2 was dephosphorylated with CIP (New England Biolabs) and purifiedwith a purification kit (Qiagen) according to the manufacturer'sinstructions. The 2.7 kbp fragment from pANN02 was ligated into thedephosphorylated 10.3 kbp linearized vector from pANN02 using T4 ligase(New England Biolabs) in a total reaction volume of 10 μL composed of 36ng of the vector, 28 ng of the insert, 1 μL 10× ligation buffer with 10mM ATP (New England Biolabs), and 1 μL T4 ligase (New England Biolabs).The reaction was incubated for 30 minutes at room temperature and a 2 μLaliquot of the reaction was transformed into electro-competent E. coliTOP10 cells (Invitrogen) according to manufacturer's instructions.Transformants were plated on LB+Kanamycin plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper insertion by colony PCR with primers oJY44 and oHJ1 (yielding aband of approximately 1.3 kbp). A clone yielding correct insertion wasdesignated pANN5.

Construction of a Right-Hand Fragment

A right-hand construct containing two B. licheniformis ADC codingregions and the I. orientalis PDC locus 3′ targeting flanking DNA wasconstructed as follows. The pMhCt071 plasmid (a plasmid identical topMhCt077 above except that the S. cerevisiae 3-HPDH ORF is not codonoptimized for I. orientalis) was digested with PmeI and PacI, treatedwith 10 units calf intestinal phosphatase (New England Biolabs), andpurified by 0.9% agarose gel electrophoresis in TAE buffer, and anapproximately 4.7 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit according to the manufacturer's instructions.

The plasmid pMeJi312-2 (supra; see FIG. 15) was digested with PmeI andPacI to extract two B. licheniformis ADC expression cassettes andpurified by 0.9% agarose gel electrophoresis in TAE buffer. Anapproximately 2.8 kbp band was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit according to the manufacturer's instructions.

The fragment containing dual B. licheniformis ADC coding regions frompMeJi312-2 was then ligated into the linearized pMhCt071 vector fragmentin a ligation reaction (20 μL) containing 1× Quick ligation buffer (NewEngland Biolabs), 1 μl 4.7 kbp fragment of pMhCt071 vector, 3 μL 2.8 kbpinsert from pMeJi312-2, and 1 μl Quick T4 DNA ligase (New EnglandBiolabs). The ligation reaction was incubated for 5 min at roomtemperature, and then the tube was placed on ice. 5 uL of this reactionwas used to transform SoloPack Gold SuperCompetent Cells (AgilentTechnologies) according to the manufacturer's instructions.Transformants were plated onto 2× YT+amp plates and incubated at 37° C.overnight. Several of the resulting transformants were screened forproper ligation of the desired fragments by ApaLI digestion. A cloneyielding the desired band sizes was kept and designated pMhCt110.

The plasmid pMhCt110 was digested with XbaI and PacI followed bytreatment with CIP and purified by 1% agarose gel electrophoresis in TBEbuffer as described herein. A band at approximately 7.1 kbp was excisedfrom the gel and purified using QIAQUICK® Gel Extraction Kit (Qiagen)according to the manufacturer's instructions.

The codon-optimized S. cerevisiae 3-HPDH coding sequence of SEQ ID NO:144 was excised from pMIBa123 (supra) by digestion with XbaI and PacIand purified by 1% agarose gel electrophoresis in TBE buffer asdescribed herein. A band at approximately 814 bp was excised from thegel and purified using QIAQUICK® Gel Extraction Kit (Qiagen) accordingto the manufacturer's instructions. The 814 bp purified fragment wasligated into the 7.1 kbp fragment from pMhCt110 using T4 ligase (NewEngland Biolabs) in a total reaction volume of 10 μL composed of 1 μL ofthe digested pMhCt110, 1 or 7 μL of the 814 bp fragment from pMIBa123, 1μL 10× ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4ligase (New England Biolabs). The reaction was incubated for 30 minutesat room temperature and a 4 μL aliquot of the reaction was transformedinto ONE SHOT® TOP10 chemically competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated on2×YT+amp plates and incubated over the weekend at room temperature.Several of the resulting transformants were screened for properinsertion by restriction digest using 2 combinations of enzymes XbaI andPacI and AscI and PacI. A clone yielding correct digested band sizesfrom each digest was designated pMIBa142.

Plasmid pMIBa142 was digested with AscI followed by a fill-in reactionwith Klenow and subsequent digestion with NheI and CIP treatment. Thedigestion was purified by 1% agarose gel electrophoresis in TBE bufferas described herein. A band at approximately 7.5 kbp was excised fromthe gel and purified using QIAQUICK® Gel Extraction Kit (Qiagen)according to the manufacturer's instructions.

The nucleotide sequence SEQ ID NO: 142 encoding the S. kluyveri BAAT ofSEQ ID NO: 21, was excised from pMIBa124 (supra) by digestion with PacIfollowed by a fill-in reaction with Klenow and subsequent digestion withXbaI. The digestion was purified by 1% agarose gel electrophoresis inTBE buffer as described herein. A band at approximately 1.4 kbp wasexcised from the gel and purified using QIAQUICK® Gel Extraction Kit(Qiagen) according to the manufacturer's instructions.

The 1.4 kbp purified fragment from pMIBa124 was ligated into the 7.5 kbpfragment from pMIBa142 using T4 ligase (New England Biolabs) in a totalreaction volume of 10 μL composed of 1 μL of the digested pMIBa142, 7 μLof the 1.4 fragment from pMIBa124, 1 μL 10× ligation buffer with 10 mMATP (New England Biolabs), and 1 μL T4 ligase (New England Biolabs). Thereaction was incubated for 2.5 hours at 16° C. and the entire ligationwas transformed into Sure cells (Agilent) according to manufacturer'sinstructions. Transformants were plated on 2× YT+amp plates andincubated overnight at 37° C. Several of the resulting transformantswere screened for proper insertion by restriction digest with StuI andPmeI. A clone yielding correct digested band size was designatedpMIBa144.

Integration of a Left-Hand and Tight-Hand Fragments into MIBa357

Plasmid pANN5 was digested with NotI and NheI and plasmid pMIBa144 wasdigested with NotI as described herein. These were purified by 1%agarose gel electrophoresis in TBE buffer, and the 8.2 kbp fragment frompANN5 and the 6 kbp fragment from pMIBa144 were excised from the gel andpurified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) accordingto manufacturer's instructions.

MIBa357 was transformed with the digested pANN5 and pMIBa144 DNA andcorrect loci targeting and transformation was verified by PCR using thePhire® Plant Direct PCR kit (Finnzymes) according to the manufacturer'sinstructions. Primers 0611552 and 0613695 yielded an approximately 4.1kbp band (to confirm the left half integration at the pdc locus);primers 0612358 and 0611554 yielded an approximately 2.5 kbp band (toconfirm the right half integration at the pdc locus); 0611245 and0611631 yielded an approximately 3.5 kbp band (to confirm the left half4×ADC integration remained at the adh1202 locus); and primers 0611815and 0611632 yielded an approximately 4.6 kbp band (to confirm the righthalf 4×ADC integration remained at the adh1202 locus). One isolate whichgave the expected bands for proper integrating of the expressioncassette at the pdc locus and retained the expression cassette at theadh1202 locus was saved and designated MIBa360.

Removal of Ura Marker from MIBa360

A ura-derivative of MIBa360 was isolated as described above. GenomicDNAs from several FOA resistant colonies of MIBa360 were screened by PCRfor the desired loop-out event with primers 0611815 and 0613689. Thepresence of an approximately 1.9 kbp band indicates the removal of theura marker with the ura3 scar site (a single URA3 promoter left behindafter homologous recombination between the two URA3 promoters in theparent strain) as desired, while a band of approximately 3.3 kbpindicates the presence of the intact URA3 promoter-URA3 ORF-URA3terminator-URA3 promoter cassette, indicating the desired recombinationevent did not occur. PCR was performed using the Phire® Plant Direct PCRkit (Finnzymes) according to the manufacturer's instructions. Two FOAresistant colonies that yielded the approximately 1.9 kbp fragment withthe above primers were saved and designated MIBa363 and MIBa364.

Construction of a Reverse Expression Cassette Right-Hand Fragment

Plasmid pMIBa144 contains the desired ADC and 3-HPDH expressioncassettes going in the forward orientation. To ease screening ofhomozygous strains, a new plasmid was constructed where the ADCexpression cassette of pMIBa144 was placed in the reverse orientation.The plasmid pMIBa144 (supra) was digested with StuI and PmeI purified by1% agarose gel electrophoresis in TBE buffer as described herein. Bandsat approximately 6.1 kbp and 2.8 kbp were excised from the gel andpurified using QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions. The 6.1 kbp fragment from pMIBa144 wastreated with CIP and purified using the QIAQUICK® PCR Purification Kit(Qiagen) according to the manufacturer's instructions.

The 2.8 kbp purified fragment from pMIBa144 was ligated into the 6.1 kbppMIBa144 linearized vector using T4 ligase (New England Biolabs) in atotal reaction volume of 10 μL composed of 1 μL of the 6.1 kbp fragmentfrom pMIBa144, 7 μL of the 2.8 kbp fragment from pMIBa144, 1 μL 10×ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4 ligase(New England Biolabs). The reaction was incubated for 4 hours at 16° C.and a 4 μL aliquot of the reaction was transformed into ONE SHOT® TOP10chemically competent E. coli cells (Invitrogen) according tomanufacturer's instructions. Transformants were plated on 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by restriction digestusing SphI and XbaI. A clone yielding correct digested band size wasdesignated pMIBa146.

Integration of a Left-Hand and Right-Hand Fragments into MIBa363

Plasmid pANN5 was digested with NotI and NheI and plasmid pMIBa146 wasdigested with NotI as described herein. These were purified by 1%agarose gel electrophoresis in TBE buffer, and the 8.2 kbp fragment frompANN5 and the 6.2 kbp fragment from pMIBa146 were excised from the geland purified the QIAQUICK® PCR Purification Kit (Qiagen) according tothe manufacturer's instructions.

MIBa363 was transformed with the digested pANN5 and pMIBa144 DNA andcorrect loci targeting and transformation was verified by PCR using thePhire® Plant Direct PCR kit (Finnzymes) or Kapa Robust DNA polymeraseaccording to the manufacturer's instructions. To confirm integrations atpdc locus, the following primer pairs were used. Primers 0613689 and0611815 yielded an approximately 1.9 kbp band; primers 0612366 and0611554 yielded an approximately 2.5 kbp band; 0613688 and 0611815yielded an approximately 3.2 kbp band; 0611622 and 0611552 yielded anapproximately 945 bp band. To check the integrations at adh1202 thefollowing primer pairs were used. Primers 0611245 and 0612794 yielded anapproximately 2.8 kbp band and primers 0611815 and 0612795 yielded anapproximately 3.9 kbp band. Two isolates which gave the expected bandsfor proper integrating of the expression cassette at the pdc locus andretained the expression cassette at the adhI202 locus were saved anddesignated MIBa372 and MIBa373.

Removal of Ura Marker from MIBa372

A ura-derivative of MIBa372 was isolated as described above. GenomicDNAs from several FOA resistant colonies of MIBa372 were screened by PCRfor the desired loop-out event with primers 0611815 and 0613688. Thepresence of an approximately 2.1 kbp band indicates the removal of theura marker with the ura3 scar site (a single URA3 promoter left behindafter homologous recombination between the two URA3 promoters in theparent strain) as desired, while a band of approximately 3.3 kbpindicates the presence of the intact URA3 promoter-URA3 ORF-URA3terminator-URA3 promoter cassette, indicating the desired recombinationevent did not occur. FOA resistant colonies of MIBA372 were alsoscreened by PCR with primers 0611815 and 0613689 (amplifies 1.9 kbpfragment) to confirm modification of the first chromosome and 0611552and 0611553 (amplifies 850 bp fragment if the pdc locus is present) toconfirm loss of the pdc locus. PCR was performed using the Phire® PlantDirect PCR kit (Finnzymes) according to the manufacturer's instructions.An FOA resistant colony that yielded the approximately 1.9 kbp fragmentwith 0611815 and 0613689, but did not amplify fragments with 0613688 and0611815, or with 0611552 and 0611553 was saved and designated MIBa375.The genotype of MIBa375 is shown in Table 28.

TABLE 28 Transformant genotype Strain Genotype MIBa375adh1202Δ::(PDC_(promo)-Opt.BlpanD PDC_(promo)-Opt.BlpanD Ura3-ScarTDH3_(promo) Opt.BlpanD TDH3_(promo)Opt.BlpanD)/adh1202Δ::(PDC_(promo)-Opt.BlpanD PDC_(promo)-Opt.BlpanDUra3-Scar [TDH3_(promo) Opt.BlpanD TDH3_(promo) Opt.BlpanD]reverse)pdcΔ::(PDC_(promo)-CNB1pyc ENO1_(promo)-CNB1aat Ura3-Scar TDH3_(promo)-OptSkPYD4 PGK1_(promo)-OptScYMR226c/pdcΔ::(PDC_(promo)-CNB1pycENO1_(promo)-CNB1aat Ura3-Scar TDH3_(promo)-OptSkPYD4 PGK1_(promo)-OptScYMR226c ura3-/ura3-

Example 3A-23: Yeast Strains Deleted for the Glycerol 3-PhosphateDehydrogenase (GPD) Gene Deletion of the GPD Gene in Strain MIBa375

Additional constructs were designed to delete both copies of glycerol3-phosphate dehydrogenase gene (SEQ ID NO: 117, which encodes GPD of SEQID NO: 118) from the host I. orientalis genome. These constructscontained approximately 1003 bp of nucleotide sequence homologous to thesequence upstream of the GPD gene and approximately 852 bp of sequencehomologous to the sequence downstream of the GPD gene, with aT_(PDC)-URA3 marker cassette (PDC terminator-URA3 promoter-URA3gene-URA3 terminator-URA3 promoter) cloned in between.

The regions upstream and downstream of GPD were amplified from I.orientalis CNB1 genomic DNA using Pfu DNA polymerase as permanufacturer's specifications. The upstream region contained an ApaIsite; this was eliminated by PCR using overlapping primers designed witha mismatch to one of the nucleotides in the ApaI recognition sequence.Primer pairs oACN48/oACN51 and oACN49/oACN50 were used to amplify thesetwo overlapping fragments for the upstream region. These PCR productswere separated on and excised from a 1% agarose gel and purified using aQiaquick Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions, and were used as template for a second round of PCR withprimers oACN48/oACN49 (having forward ApaI/reverse NotI sites). Thedownstream region was amplified with primers oACN52/oACN53 (havingforward NotI/reverse SacI sites) from I. orientalis genomic DNA usingPfu DNA polymerase as per manufacturer's specifications. Both PCRproducts were gel purified and cloned separately into the vectorpCR®-BluntII-TOPO® (Invitrogen). Isolates were confirmed to have thedesired insert by restriction digest of plasmid DNA, and were verifiedby sequencing. Vector pACN58 contained the cloned upstream fragment andpACN59 contained the downstream fragment. Plasmid pACN58 was digestedwith ApaI/NotI to release the upstream flank, plasmid pACN59 wasdigested with SacI/NotI to release the downstream flank, and pACN59 wasdigested with ApaI/SacI to provide the vector backbone. The threedesired fragments were separated on a 1% agarose gel, excised andpurified, and ligated in a 3-piece ligation reaction using T4 ligase(New England Biolabs). The ligation reaction was transformed into E.coli TOP10 electrocompetent cells and transformants were confirmed byrestriction digest of plasmid DNA. During this procedure, an additionalSacI site in the downstream region was detected, which resulted in adownstream region of 853 bp (as opposed to 1 kbp). Two isolates with thedesired inserts were named pACN62 and pACN63.

The T_(PDC1)-URA3 cassette was isolated from vector pJLJ8 (FIG. 32)using a NotI digest and gel purification. This fragment was then ligatedinto pACN62 (supra) that had been digested with NotI anddephosphorylated, and the ligation was transformed into E. coli DH10Belectrocompetent cells (Invitrogen). Colonies with the URA3 insert wereconfirmed by PCR using primers oACN48/oJLJ44 and oACN48/oJLJ46. PrimeroJLJ44 anneals at the end of the downstream URA3 promoter and amplifiesoutward from the T_(PDC1)-URA3 cassette. Primer oJLJ46 anneals at the 5′end of the PDC terminator and amplifies outward from the T_(PDC1)-URA3cassette. Vector pHJJ56 contains the URA3 facing the downstream GPDregion and pHJJ57 contains the URA3 facing the upstream GPD region.

Plasmids pHJJ56 and pHJJ57 were linearized by digestion with KpnI andApaI and the fragments containing the deletion cassette were purified bygel extraction. Linearized pHJJ56 was transformed into the ura-strainMIBa375. Single colonies were restreaked for purification and tested byPCR for the desired GPD deletion using primers oJLJ44, oJLJ46, oACN54and oACN55. Cells were lysed in 40 uL Y-Lysis buffer and 2 uL Zymolyase(ZymoResearch) at 37° C. for 30 minutes and 1 uL of the lysis reactionused in a 25 uL PCR reaction. PCR reactions used Failsafe DNA polymeraseand Buffer E according to manufacturer's specifications, with anannealing temperature of 55° C. and the following cycling profile: 1cycle at 94° C. for 2 minutes; 29 cycles each at 94° C. for 30 seconds,55° C. for 30 seconds, and 72° C. for 1.5 minutes; and 1 cycle at 72° C.for 3 minutes. Strains with one copy of the GPD knockout produced bandsof approximately 0.9 and 1.2 kbp and were named yHJN1 and yHJN2.

Strains yHJN1 and yHJN2 were grown overnight in YPD media and platedonto ScD-2×FOA media to select to loss of the URA3 marker. Singlecolonies were purified on YPD and patched to ScD-ura and YPD media toconfirm the ura-phenotype. Ura-colonies were confirmed to have retainedthe knockout using the same PCR reaction used to confirm the firstintegration. A ura-derivative of yHJN1 was named yHJN3 and aura-derivative of yHJN2 was named yHJN4.

Linearized pHJJ57 was transformed into yHJN3 and yHJN4 and singlecolonies were purified on ScD-ura media. The presence of two copies ofthe GPD knockout was confirmed by PCR using primers oJLJ44, oACN54, andoACN55 in one reaction, and primers oJLJ46, oACN54, and oACN55 in asecond reaction. Primer oACN54 anneals to a region approximately 37 bpupstream of the upstream flanking sequence for GPD, while oACN55 annealsto a region approximately 24 bp downstream of the downstream flank. Theformer reaction produces bands of approximately 900 and 1050 bp if bothcopies of the GPD are deleted, and the latter reaction produces bands ofapproximately 1025 and 1200 bp. Colonies with two copies of the GPDknockout grew more slowly on ScD-ura plates than those with a singlecopy of the deletion. Strains having both copies of the GPD gene deletedwere named yHJN7 (derived from yHJN3) and yHJN8 (derived from yHJN4).

Strains MIBa372, yHJN7 and yHJN8 were tested in bioreactors for glycerolproduction, using the methods described herein. Strain MIBa372 produced29.5 g/L glycerol in 48 hours. No detectable glycerol was produced bystrains yHJN7 or yHJN8 during the fermentation. The absence of glycerolin the final fermentation broth may provide advantages in the recoveryand purification of 3-HP from the fermentation broth.

3A-24: Yeast Strains Expressing Aspartate 1-Decarboxylase (ADC) fromFour Nucleotide Sequences at the Adh1202 Locus, 3-HP Dehydrogenase(3-HPDH) at the Adh9091 Locus and Deletion of Native 3-HP Dehydrogenase(3-HPDH)

Plasmid Construction for Integration of I. orientalis 3-HPDH at theAdh9091 Locus

The nucleotide sequences of SEQ ID NO: 25 encoding the I. orientalis3-HPDH of SEQ ID NO: 26 and SEQ ID NO: 19 encoding the I. orientalisBAAT (PYD4) of SEQ ID NO: 20 were amplified from MBin500 I. orientalisgenomic DNA prepared as described previously using primers 0611954 and0611957 (for 3-HPDH) or 0611997 and 0611998 (PYD4). Primer 0611954 addsa kozak sequence (TAAA) and NheI site to the 5′ end, and primer 0611957adds a PacI site to the 3′ end of 3-HPDH during amplification. Primer0611997 adds a kozak sequence (TAAA) and PacI site to the 5′ end, andprimer 0611998 adds a PacI site to the 3′ end of PYD4 duringamplification. Fifty pmoles of each primer was used in a PCR reactioncontaining 50 ng of MBin500 genomic DNA as template, 0.2 mM each dATP,dGTP, dCTP, dTTP, 1× Expand High Fidelity Buffer (Roche), and 2.6 unitsof Expand High Fidelity DNA Polymerase (Roche) in a final volume of 50μL. The PCR was performed in an EPPENDORF® MASTERCYCLER® (EppendorfScientific) programmed for one cycle at 95° C. for 3 minutes followed by30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C.for 1 minute (3-HPDH PCR) or 2 minutes (PYD4 PCR), with a finalextension at 72° C. for 5 minutes. Following thermocycling, the PCRreaction products were separated by 1.0% agarose gel electrophoresis inTAE buffer where an approximately 831 bp 3-HPDH or 1.4 kbp PYD4 PCRproduct was excised from the gel and purified using a using a QIAQUICK®Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions. Five μl of the purified 3-HPDH or PYD4 was cloned intopCR2.1 (Invitrogen) using a TOPO-TA Cloning Kit (Invitrogen) accordingto the manufacturer's instructions. The transformations were plated onto2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened by digestion with EcoRI. Clonesyielding the desired insert size were confirmed to be correct by DNAsequencing. One clone containing 3-HPDH was designated pMBin190, andanother containing PYD4 was designated pMBin193.

Plasmid pMBin193 was digested with XbaI and PacI and run on a 1.0%agarose gel in TAE buffer where the 1.4 kbp PYD4 band was excised fromthe gel and purified using a QIAQUICK® Gel Extraction Kit (Qiagen)according to the manufacturer's instructions. The digested PYD4 fragmentwas ligated into the XbaI and PacI restricted linear pMIBa107 plasmid(supra) using T4 DNA ligase. The ligation product was transformed intoOne Shot® TOP10 Chemically Competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated onto2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened by digestion with SnaBI or EcoRI.One clone yielding the desired band sizes was designated pMBin203.Plasmid pMBin203 contains NotI sites that flank the following expressioncassette: PDC promoter and terminator up and downstream of the PYD4 CDS,the URA3 promoter, the URA3 ORF, and the URA3 terminator followed by theURA3 promoter.

Plasmid pMBin203 was digested with NotI and separated on a 1.0% agarosegel in TAE buffer where the approximately 4.1 kbp fragment (containingthe PDC promoter, PYD4 CDS, the PDC terminator, and the URA3 selectionmarker) was excised from the gel and purified using a QIAQUICK® GelExtraction Kit (Qiagen) according to the manufacturer's instructions.Plasmid pHJJ27 (containing 5′ and 3′ homology regions to the adh9091locus; see FIG. 21) was digested with NotI, treated with CIP andseparated on a 1.0% agarose gel in TAE buffer where the approximately5.7 kbp linear plasmid was purified as described above. The fragmentfrom pMBin203 was then ligated into the NotI restricted pHJJ27 using T4DNA ligase as described above. The ligation product was transformed intoOne Shot® TOP10 Chemically Competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated onto2× YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened by digestion with PstI. One doneyielding the desired band sizes was designated pMBin204. PlasmidpMBin204 allows targeting of PYD4 to the adh9091 locus.

The nucleotide sequence of SEQ ID NO: 25 encoding the I. orientalis3-HPDH of SEQ ID NO: 26 was removed from plasmid pMBin190 (supra) bydigestion with NheI and PacI and purified by agarose gel electrophoresisin TBE buffer as described herein. A band of approximately 827 bp wasexcised from the gel and purified using a NUCLEOSPIN® Extract II Kit(Macherey-Nagel) according to the manufacturer's instructions. Theplasmid pMBin204 (supra) was digested with XbaI and PacI and purified byagarose gel electrophoresis in TBE buffer as described herein. A band ofapproximately 8.4 kbp was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The purified approximately 827 bp I. orientalis 3-HPDH gene productabove was ligated into the 8.4 kbp pMBin204 linearized vector using T4ligase (New England Biolabs) in a total reaction volume of 20 μLcomposed of 1 μL of the 8.4 kbp vector, 10 μL of the 827 bp insert, 2 μL10× ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4ligase (New England Biolabs). The reaction was incubated for 18 hours at16° C. and a 10 μL aliquot of the reaction was transformed into One ShotTOP10 cells (Invitrogen) according to manufacturer's instructions. Aftera recovery period, two 100 μL aliquots from the transformation reactionwere plated onto 150 mm 2× YT plates supplemented with 100 μg ofampicillin per ml. The plates were incubated overnight at 37° C.Putative recombinant clones were selected from the selection plates andplasmid DNA was prepared from each one using a BIOROBOT® 9600 (Qiagen).Clones were analyzed by restriction digest and a plasmid with thecorrect restriction digest pattern was designated pMcTs90.

Plasmid Construction for Integration of S. cerevisiae 3-HPDH at theAdh9091 Locus

The 826 bp wild-type nucleotide sequence encoding the S. cerevisiae3-HPDH of SEQ ID NO: 129 was PCR amplified from JGI69 genomic DNA andamended with an XbaI site on the 5′ end of the gene and a PacI site onthe 3′ end of the gene. The amplification reaction was performed usingPlatinum® Pfx DNA polymerase (InVitrogen) according to manufacturer'sinstructions. A Master PCR reaction containing 1.125 ul of S. cerevisiaegenomic DNA, 112.5 μM each of primers 611191 and 611199, 1× Pfxamplification buffer (InVitrogen), 2 mm MgSO₄, 0.2 mM dNTP mix, 5 UnitsPlatinum® Pfx DNA polymerase (InVitrogen) in a final volume of 200 μL.The mix was aliquoted into eight tubes and gradient PCR performed. Theamplification reactions were incubated in an EPPENDORF® MASTERCYCLER®(Eppendorf Scientific Inc.) programmed for 1 cycle at 95° C. for 2minutes; 30 cycles each at 95° C. for 30 seconds, Gradient 40-55° C. for30 seconds, and 72° C. for 2 minutes; and 1 cycle at 72° C. for 3minutes.

The 826 bp wild-type YMR226c PCR gene product was purified by 1% agarosegel electrophoresis in TBE buffer as described herein. A fragment ofapproximately 826 bp was excised from the gel and extracted from theagarose using a QIAQUICK® Gel Extraction Kit (Qiagen). The PCR productwas digested overnight at 37° C. with XbaI and PacI then purified usingthe QIAQUICK® PCR purification Kit (Qiagen).

The plasmid pMIBa100 (supra) was digested with XbaI and PacI followed bytreatment with CIP resulting in an approximately 6.8 kbp linearfragment. The digestion was purified using the QIAQUICK® PCRpurification Kit (Qiagen) according to the manufacturer's instructions.

The 826 bp YMR226c purified and digested PCR fragment was ligated intothe 6.8 kbp pMIBa100 linearized vector using T4 ligase (New EnglandBiolabs) in a total reaction volume of 10 μL composed of 1 μL of the 6.7kbp fragment from pMIBa100, 1 μL or 7 μL of the 826 bp YMR226c PCRproduct, 1 μL 10× ligation buffer with 10 mM ATP (New England Biolabs),and 1 μL T4 ligase (New England Biolabs). The reaction was incubated forapproximately 4 hours at 16° C. and the entire reaction was transformedinto Sure chemically competent cells (Aglient) according tomanufacturer's instructions. Transformants were plated on 2× YT+ampplates and incubated at 37° C. overnight. Several of the resultingtransformants were screened for proper insertion by restriction digestusing XbaI and PacI. Correct clones by digest were confirmed by DNAsequencing. A clone yielding correct digested band size and DNA sequencewas designated pMIBa101.

Plasmid pHJJ76-no ura (supra) was digested with NotI followed bytreatment with CIP. The linear 5.2 kbp fragment was purified using aQIAQUICK® PCR Purification Kit (Qiagen).

The YMR226c expression cassette was excised from pMIBa101 by digestionwith NotI. A band at approximately 3546 bp was excised from the gel andpurified using QIAQUICK® Gel Extraction Kit (Qiagen) according to themanufacturer's instructions.

The 3546 bp purified fragment from pMIBa101 was ligated into the 5.2 kbppHJJ76-no ura linearized vector using T4 ligase (New England Biolabs) ina total reaction volume of 10 μL composed of 1 μL of the 5.2 kbpfragment from pHJJ76-no ura, 1 μL or 5 μL of the 3546 bp fragment frompMIBa101, 1 μL 10× ligation buffer with 10 mM ATP (New England Biolabs),and 1 μL T4 ligase (New England Biolabs). The reaction was incubatedovernight at 16° C. and a 4 μL aliquot of the reaction was transformedinto ONE SHOT® TOP10 chemically competent E. coli cells (Invitrogen)according to manufacturer's instructions. Transformants were plated on2×YT+amp plates and incubated at 37° C. overnight. Several of theresulting transformants were screened for proper insertion byrestriction digest using XbaI and KpnI. A clone yielding correctdigested band size was designated pMIBa109.

The wild-type nucleotide sequence encoding the S. cerevisiae 3-HPDH ofSEQ ID NO: 129 was removed from plasmid pMIBa109 by digestion with XbaIand PacI and purified by agarose gel electrophoresis in TBE buffer asdescribed herein. A band of approximately 818 bp was excised from thegel and purified using a NUCLEOSPIN® Extract II Kit (Macherey-Nagel)according to the manufacturer's instructions.

The purified approximately 818 bp S. cerevisiae 3-HPDH gene product wasligated into the 8.4 kbp pMBin204 linearized vector above using T4ligase (New England Biolabs) in a total reaction volume of 20 μLcomposed of 1 μL of the 8.4 kbp vector, 10 μL of the 818 bp insert, 2 μL10× ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4ligase (New England Biolabs). The reaction was incubated for 18 hours at16° C. and a 10 μL aliquot of the reaction was transformed into One ShotTOP10 cells (Invitrogen) according to manufacturer's instructions. Aftera recovery period, two 100 μL aliquots from the transformation reactionwere plated onto 150 mm 2× YT plates supplemented with 100 μg ofampicillin per mL. The plates were incubated overnight at 37° C.Putative recombinant clones were selected from the selection plates andplasmid DNA was prepared from each one using a BIOROBOT® 9600 (Qiagen).Clones were analyzed by restriction digest and a plasmid with thecorrect restriction digest pattern was designated pMcTs91.

Plasmid Construction for Integration of M. sedula 3-HPDH at the Adh9091Locus

An I. orientalis codon-optimized nucleotide sequence of SEQ ID NO: 343encoding the M. sedula 3-HPDH of SEQ ID NO: 29 was synthesized byGeneArt® resulting in the plasmid 11AAE2AP. The synthetic gene wasdigested from the plasmid with XbaI and PacI and purified by agarose gelelectrophoresis in TBE buffer as described herein. A band ofapproximately 959 bp was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The purified approximately 959 bp M. sedula 3-HPDH gene product wasligated into the 8.4 kbp pMBin204 linearized vector above using T4ligase (New England Biolabs) in a total reaction volume of 20 μLcomposed of 1 μl of the 8.4 kbp vector, 16 μl of the 959 bp insert, 2 μL10× ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4ligase (New England Biolabs). The reaction was incubated for 1 hour atroom temperature and a 10 μL aliquot of the reaction was transformedinto Solo Pack Gold Super Competent cells (Agilent) according tomanufacturer's instructions. After a recovery period, two 100 μlaliquots from the transformation reaction were plated onto 150 mm 2× YTplates supplemented with 100 μg of ampicillin per ml. The plates wereincubated overnight at 37° C. Putative recombinant clones were selectedfrom the selection plates and plasmid DNA was prepared from each oneusing a BIOROBOT® 9600 (Qiagen). Clones were analyzed by restrictiondigest and a plasmid with the correct restriction digest pattern wasdesignated pMcTs76.

Plasmid Construction for Integration of E. coli 3-HPDH at Adh9091 Locus

An I. orientalis codon-optimized nucleotide sequence of SEQ ID NO: 143encoding the E. coli 3-HPDH of SEQ ID NO: 27 was synthesized by GeneArt®resulting in the plasmid 1045168. The synthetic gene was digested fromthe plasmid with XbaI and PacI and purified by agarose gelelectrophoresis in TBE buffer as described herein. A band ofapproximately 761 bp was excised from the gel and purified using aNUCLEOSPIN® Extract II Kit (Macherey-Nagel) according to themanufacturer's instructions.

The purified approximately 761 bp E. coli 3-HPDH gene product wasligated into the 8.4 kbp pMBin204 linearized vector above using T4ligase (New England Biolabs) in a total reaction volume of 20 μLcomposed of 1 μL of the 8.4 kbp vector, 16 μL of the 761 bp insert, 2 μL10× ligation buffer with 10 mM ATP (New England Biolabs), and 1 μL T4ligase (New England Biolabs). The reaction was incubated for 1 hr atroom temperature and a 10 μL aliquot of the reaction was transformedinto Solo Pack Gold Super Competent cells (Agilent) according tomanufacturer's instructions. After a recovery period, two 100 μLaliquots from the transformation reaction were plated onto 150 mm 2× YTplates supplemented with 100 μg of ampicillin per ml. The plates wereincubated overnight at 37° C. Putative recombinant clones were selectedfrom the selection plates and plasmid DNA was prepared from each oneusing a BIOROBOT® 9600 (Qiagen). Clones were analyzed by restrictiondigest and a plasmid with the correct restriction digest pattern wasdesignated pMcTs77.

Integration of 3-HPDH Fragments at Adh9091 in McTs259

Plasmids pMcTs76, pMcTs77, pMcTs91 were digested with KpnI and ApaI, andplasmid pMcTs90 was digested with SacI and ApaI as described herein. Theresulting digestion products were purified by 1% agarose gelelectrophoresis in TBE buffer, and the 5.5 kbp fragment from pMcTs76,the 5.3 kbp fragment from pMcTs77, the 5.4 kbp fragment from pMcTs90,and 5.4 kbp fragment from pMcTs91 were excised from the gel and purifiedusing a NUCLEOSPIN® Extract II Kit (Macherey-Nagel) the according to themanufacturer's instructions.

Strain McTs259 which expressing four copies of nucleotides encoding theB. licheniformis ADC of SEQ ID NO: 139 at the adh1202 locus and deletionof the native I. orientalis gene encoding the 3-HPDH of SEQ ID NO: 26(supra), was transformed with the digested pMcTs76, pMcTs77, pMcTs90, orpMcTs91 DNA. The correct loci targeting and transformation was verifiedby PCR using the Phire® Plant Direct PCR kit (Finnzymes) according tothe manufacturer's instructions. To confirm integrations at adh9091locus, the following primer pairs were used. Primers 0614627 and 0612909yielded an approximately 3.47 kbp band for fragment from pMcTs76integrated, approximately 3.27 kbp band for fragment from pMcTs77integrated, approximately 3.34 kbp band for fragment from pMcTs90integrated, approximately 3.33 kbp band for fragment from pMcTs91integrated; primers 0612908 and 0614626 yielded an approximately 1.97kbp band. To check the integrations at adh1202 the following primerpairs were used. Primers 0611245 and 0612794 yielded an approximately3.0 kbp band and primers 0611815 and 0612795 yielded an approximately3.6 kbp band. To check the deletion of the native I. orientalis 3-HPDHgene the following primers were used. Primers 0613034 and 0613241yielded an approximately 1.4 kbp band. Isolates which gave the expectedbands for proper integrating of the expression cassette at the adh9091locus, retained the expression cassette at the adh1202 and retained thedeletion I. orientalis 3-HPDH locus were saved and designated McTs261(pMcTs76 fragment), McTs263 (pMcTs77 fragment), McTs267 (pMcTs90fragment), and McTs269 (pMcTs91 fragment) as shown in Table 29.

TABLE 29 Transformant constructs Construction Gene Gene ProductIntegration Plasmid Gene Source SEQ ID NO construct TransformantpMBin190 3-HPDH I. orientalis 26 pMcTs90 McTs267 (YMR226c) 11AAE2AP3-HPDH M. sedula 29 pMcTs76 McTs261 (Msed_1993) pMIBa109 3-HPDH S.cerevisiae 129 pMcTs91 McTs269 (YMR226c) 1045168 3-HPDH E. coli 27pMcTs77 McTs263 (ydfG)

The transformant strains were tested for 3-HP production using the shakeflask method described above. The heterozygous transformants McTs267,McTs269, and McTs263 produced 0.149 (+/−0.024), 0.168 (+/−0.052), and0.162 (+/−0.018) g/L 3-HP per g/L dry cell weight, respectively. Nativestrain MeJi412 produced 0.263 (+/−0.026) g/L 3-HP per g/L dry cellweight, and the 3-HPDH deletion strain produced no detectable 3-HP. Theheterozygous transformant McTs261 did not produce detectable 3-HP withthis experiment. These results suggest that even heterozygous 3-HPDHtransformants can restore some 3-HPDH activity of 3-HPDH deletion strainusing either exogenous or endogenous 3-HPDH gene sequences.

Example 3B: Modified Yeast Strains Expressing Malate Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes PEP, OAA, andmalate intermediates can be engineered by expressing one or more enzymesinvolved in the pathway. The expressed genes may include one or more ofa PPC, malate dehydrogenase, and malate decarboxylase gene. Theexpressed genes may be derived from a gene that is native to the hostcell, or they may be derived from a source gene that is non-native tothe host cell.

Example 3C: Modified Yeast Strains Expressing Malonate SemialdehydePathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes PEP, OAA andmalonate semialdehyde intermediates can be engineered by expressing oneor more enzymes involved in the pathway. The expressed genes may includeone or more of a PPC, 2-keto acid decarboxylase, KGD, BCKA,indolepyruvate decarboxylase, 3-HPDH (including malonate semialdehydereductase), HIBADH, and 4-hydroxybutyrate dehydrogenase gene. Theexpressed genes may be derived from a gene that is native to the hostcell, or they may be derived from a source gene that is non-native tothe host cell.

Example 3D: Modified Yeast Strains Expressing Malonyl-CoA Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes PEP, OAA,malonyl-CoA, and, optionally, malonate semialdehyde intermediates can beengineered by expressing one or more enzymes involved in the pathway.The expressed genes may include one or more of a PPC, OAA formatelyase,malonyl-CoA reductase, CoA acylating malonate semialdehydedehydrogenase, 3-HPDH (including malonate semialdehyde reductase),HIBADH, and 4-hydroxybutyrate dehydrogenase gene. The expressed genesmay be derived from a gene that is native to the host cell, or they maybe derived from a source gene that is non-native to the host cell.

Example 3E: Modified Yeast Strains Expressing Malonyl-CoA Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes pyruvate,acetyl-CoA, malonyl-CoA, and, optionally, malonate semialdehydeintermediates can be engineered by expressing one or more enzymesinvolved in the pathway. The expressed genes may include one or more ofa PDH, acetyl-CoA carboxylase, malonyl-CoA reductase, CoA acylatingmalonate semialdehyde dehydrogenase, 3-HPDH (including malonatesemialdehyde reductase), HIBADH, and 4-hydroxybutyrate dehydrogenasegene. The expressed genes may be derived from a gene that is native tothe host cell, or they may be derived from a source gene that isnon-native to the host cell.

Example 3F: Modified Yeast Strains Expressing Alanine Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes pyruvate,alanine, β-alanine, and, optionally, malonate semialdehyde,β-alanyl-CoA, acrylyl-CoA, and 3-HP-CoA intermediates can be engineeredby expressing one or more enzymes involved in the pathway. The expressedgenes may include one or more of an alanine dehydrogenase,pyruvate/alanine aminotransferase, alanine 2,3 aminomutase, CoAtransferase, CoA synthetase, β-alanyl-CoA ammonia lyase, 3-HP-CoAdehydratase, 3-HP-CoA hydrolase, 3-hydroxyisobutyryl-CoA hydrolase,BAAT, 3-HPDH (including malonate semialdehyde reductase), HIBADH, and4-hydroxybutyrate dehydrogenase gene. The expressed genes may be derivedfrom a gene that is native to the host cell, or they may be derived froma source gene that is non-native to the host cell.

Example 3G: Modified Yeast Strains Expressing Lactate Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes pyruvate,lactate, lactyl-CoA, acrylyl-CoA, and 3-HP-CoA intermediates can beengineered by expressing one or more enzymes involved in this pathway.The expressed genes may include one or more of an LDH, CoA transferase,CoA synthetase, lactyl-CoA dehydratase, 3-HP-CoA dehydratase, 3-HP-CoAhydrolase, and 3-hydroxyisobutyryl-CoA hydrolase gene. The expressedgenes may be derived from a gene that is native to the host cell, orthey may be derived from a source gene that is non-native to the hostcell.

Example 3H: Modified Yeast Strains Expressing Glycerol Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes glycerol and3-HPA intermediates can be engineered by expressing one or more enzymesinvolved in this pathway. The expressed genes may include one or more ofa glycerol dehydratase and aldehyde dehydrogenase gene. The expressedgenes may be derived from a gene that is native to the host cell, orthey may be derived from a source gene that is non-native to the hostcell.

Example 31: Modified Yeast Strains Expressing β-Alanyl CoA Pathway Genes

Yeast cells that produce 3-HP via a pathway that utilizes PEP orpyruvate, β-alanine, β-alanyl-CoA, acrylyl-CoA, 3-HP-CoA, and,optionally OAA, aspartate, and alanine intermediates can be engineeredby expressing one or more enzymes involved in this pathway. Theexpressed genes may include one or more of a PPC, PYC, AAT, ADC, CoAtransferase, CoA synthetase, β-alanyl-CoA ammonia lyase, 3-HP-CoAdehydratase, 3-HP-CoA hydrolase, 3-hydroxyisobutyrl-CoA hydrolase,alanine dehydrogenase, pyruvate/alanine aminotransferase, and AAM gene.The expressed genes may be derived from a gene that is native to thehost cell, or they may be derived from a source gene that is non-nativeto the host cell.

In some aspects, the yeast cells or methods of use thereof may bedescribed by the following numbered paragraphs:

[B1] A genetically modified yeast cell comprising an active 3-HPfermentation pathway, wherein the cell comprises one or more exogenous3-HP pathway genes selected from:

-   -   an exogenous PPC gene;    -   an exogenous PYC gene;    -   an exogenous AAT gene;    -   an exogenous ADC gene;    -   an exogenous BAAT or gabT gene; and    -   an exogenous 3-HPDH gene.        [B2] The genetically modified yeast cell of paragraph B1,        comprising an exogenous AAT gene.        [B3] The genetically modified yeast cell of paragraph B1 or B2,        comprising an exogenous PYC gene.        [B4] The genetically modified yeast cell of any of paragraphs        B1-B3, comprising an exogenous ADC gene.        [B5] The genetically modified yeast cell of any of paragraphs        B1-B4, comprising an exogenous BAAT or gabT gene.        [B6] The genetically modified yeast cell of any of paragraphs        B1-B5, comprising an exogenous 3-HPDH gene.        [B7] The genetically modified yeast cell of paragraph B1,        comprising:    -   an exogenous PYC gene;    -   an exogenous AAT gene;    -   an exogenous ADC gene;    -   an exogenous BAAT or gabT gene; and    -   an exogenous 3-HPDH gene.        [B8] The genetically modified yeast cell of any of paragraphs        B1-B7, comprising an exogenous PPC gene.        [B9] The genetically modified yeast cell of any of paragraphs        B1-B7, wherein the exogenous PYC gene encodes a polypeptide with        at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to an amino acid        sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8.        [B10] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to an amino acid        sequence selected from SEQ ID NOs: 2.        [B11] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 3.        [B12] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 4.        [B13] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 5.        [B14] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 6.        [B15] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 7.        [B16] The genetically modified yeast cell of paragraph B9,        wherein the exogenous PYC gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 8.        [B17] The genetically modified yeast cell of any of paragraphs        B1-B16, wherein the exogenous PYC gene comprises a nucleotide        sequence with at least 60%, e.g., at least 65%, 70%, 75%, 80%,        85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the        nucleotide sequence of SEQ ID NO: 1.        [B18] The genetically modified yeast cell of any of paragraphs        B1-B17, wherein the AAT gene encodes a polypeptide with at least        60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,        99%, or 100% sequence identity to an amino acid sequence        selected from SEQ ID NOs: 14, 15, and 16.        [B19] The genetically modified yeast cell of paragraph B18,        wherein the AAT gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 14.        [B20] The genetically modified yeast cell of paragraph B18,        wherein the AAT gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 15.        [B21] The genetically modified yeast cell of paragraph B18,        wherein the AAT gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 16.        [B22] The genetically modified yeast cell of any of paragraphs        B1-B21, wherein the AAT gene comprises a nucleotide sequence        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 13.        [B23] The genetically modified yeast cell of any of paragraphs        B1-B22, wherein the ADC gene encodes a polypeptide with at least        60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,        99%, or 100% sequence identity to an amino acid sequence        selected from SEQ ID NOs: 17, 18, 133, 135, 137, and 139.        [B24] The genetically modified yeast cell of paragraph B23,        wherein the ADC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 17.        [B25] The genetically modified yeast cell of paragraph B23,        wherein the ADC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 18.        [B26] The genetically modified yeast cell of paragraph B23,        wherein the ADC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 133.        [B27] The genetically modified yeast cell of paragraph B23,        wherein the ADC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 135.        [B28] The genetically modified yeast cell of paragraph B23,        wherein the ADC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 137.        [B29] The genetically modified yeast cell of paragraph B23,        wherein the ADC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 139.        [B30] The genetically modified yeast cell of any of paragraphs        B1-B29, wherein the ADC gene comprises a nucleotide sequence        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide        sequence selected from SEQ ID NOs: 130, 131, 132, 134, 136, and        138.        [B31] The genetically modified yeast cell of paragraph B30,        wherein the ADC gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 130.        [B32] The genetically modified yeast cell of paragraph B30,        wherein the ADC gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 131.        [B33] The genetically modified yeast cell of paragraph B30,        wherein the ADC gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 132.        [B34] The genetically modified yeast cell of paragraph B30,        wherein the ADC gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nudeotide        sequence of SEQ ID NO: 134.        [B35] The genetically modified yeast cell of paragraph B30,        wherein the ADC gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 136.        [B36] The genetically modified yeast cell of paragraph B30,        wherein the ADC gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 138.        [B37] The genetically modified yeast cell of any of paragraphs        B1-B36, wherein the exogenous BAAT or gabT gene encodes a        polypeptide with at least 60%, e.g., at least 65%, 70%, 75%,        80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to        an amino acid sequence selected from SEQ ID NOs: 20, 21, 22, 23,        and 24.        [B38] The genetically modified yeast cell of paragraph B37,        wherein the exogenous BAAT or gabT gene encodes a polypeptide        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 20.        [B39] The genetically modified yeast cell of paragraph B37,        wherein the exogenous BAAT or gabT gene encodes a polypeptide        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 21.        [B40] The genetically modified yeast cell of paragraph B37,        wherein the exogenous BAAT or gabT gene encodes a polypeptide        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 22.        [B41] The genetically modified yeast cell of paragraph B37,        wherein the exogenous BAAT or gabT gene encodes a polypeptide        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 23.        [B42] The genetically modified yeast cell of paragraph B37,        wherein the exogenous BAAT or gabT gene encodes a polypeptide        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid        sequence of SEQ ID NO: 24.        [B43] The genetically modified yeast cell any of paragraphs        B1-B42, wherein the BAAT gene or gabT gene comprises a        nucleotide sequence with at least 60%, e.g., at least 65%, 70%,        75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence        identity to a nucleotide sequence selected from SEQ ID NOs: 19,        140, 141, and 142.        [B44] The genetically modified yeast cell any of paragraph B43,        wherein the BAAT gene or gabT gene comprises a nucleotide        sequence with at least 60%, e.g., at least 65%, 70%, 75%, 80%,        85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the        nucleotide sequence of SEQ ID NO: 19.        [B45] The genetically modified yeast cell any of paragraph B43,        wherein the BAAT gene or gabT gene comprises a nucleotide        sequence with at least 60%, e.g., at least 65%, 70%, 75%, 80%,        85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the        nucleotide sequence of SEQ ID NO: 140.        [B46] The genetically modified yeast cell any of paragraph B43,        wherein the BAAT gene or gabT gene comprises a nucleotide        sequence with at least 60%, e.g., at least 65%, 70%, 75%, 80%,        85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the        nucleotide sequence of SEQ ID NO: 141.        [B47] The genetically modified yeast cell any of paragraph B43,        wherein the BAAT gene or gabT gene comprises a nucleotide        sequence with at least 60%, e.g., at least 65%, 70%, 75%, 80%,        85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the        nucleotide sequence of SEQ ID NO: 142.        [B48] The genetically modified yeast cell of any of paragraphs        B1-B47, wherein said exogenous BAAT gene or exogenous gabT is a        BAAT gene that is also a gabT gene.        [B49] The genetically modified yeast cell of any of paragraphs        B1-B48, wherein the 3-HPDH gene encodes a polypeptide with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 26,        27, 28, 29, 30, 31, 32, 33, 34, and 129.        [B50] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 26.        [B51] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 27.        [B52] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 28.        [B53] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 29.        [B54] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 30.        [B55] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 31.        [B56] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 32.        [B57] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 33.        [B58] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 34.        [B59] The genetically modified yeast cell of paragraph B49,        wherein the 3-HPDH gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 129.        [B60] The genetically modified yeast cell of any of paragraphs        B1-B59, wherein the 3-HPDH gene comprises a nucleotide sequence        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide        sequence selected from SEQ ID NOs: 25, 143, 144, and 343.        [B61] The genetically modified yeast cell of paragraph B60,        wherein the 3-HPDH gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 25.        [B62] The genetically modified yeast cell of paragraph B60,        wherein the 3-HPDH gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 143.        [B63] The genetically modified yeast cell of paragraph B60,        wherein the 3-HPDH gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 144.        [B64] The genetically modified yeast cell of paragraph B60,        wherein the 3-HPDH gene comprises a nucleotide sequence with at        least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,        97%, 98%, 99%, or 100% sequence identity to the nucleotide        sequence of SEQ ID NO: 343.        [B65] The genetically modified yeast cell of any of paragraphs        B1-B64, wherein the 3-HPDH gene is also a HIBADH gene.        [B66] The genetically modified yeast cell of any of paragraphs        B1-B65, wherein the 3-HPDH gene is also a 4-hydroxybutyrate        dehydrogenase gene.        [B67] The genetically modified yeast cell of any of paragraphs        B1-B66, wherein the PPC gene encodes a polypeptide with at least        60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,        99%, or 100% sequence identity to an amino acid sequence        selected from SEQ ID NOs: 10, 11, and 12.        [B68] The genetically modified yeast cell of paragraph 867,        wherein the PPC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 10.        [B69] The genetically modified yeast cell of paragraph B67,        wherein the PPC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 11.        [B70] The genetically modified yeast cell of paragraph B67,        wherein the PPC gene encodes a polypeptide with at least 60%,        e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,        or 100% sequence identity to the amino acid sequence of SEQ ID        NO: 12.        [B71] The genetically modified yeast cell of any of paragraphs        B1-870, wherein the PPC gene comprises a nucleotide sequence        with at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%,        95%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide        sequence of SEQ ID NO: 9.        [B72] The genetically modified yeast cell of any of paragraphs        B1-B71, wherein said yeast cell is Crabtree-negative.        [B73] The genetically modified yeast cell of any of paragraphs        B1-B72, wherein the yeast cell belongs to a genus selected from        Issatchenkia, Candida, Kluyveromyces, Pichia,        Schizosaccharomyces, Torulaspora, Zygosaccharomyces, and        Saccharomyces.        [B74] The genetically modified yeast cell of paragraph B73,        wherein the yeast cell belongs to a clade selected from the I.        orientalis/P. fermentans clade and the Saccharomyces clade.        [B75] The genetically modified yeast cell of paragraph B73,        wherein the yeast cell is selected from I. orientalis, C.        lambica, and S. bulderi.        [B76] The genetically modified yeast cell of any of paragraphs        B1-B75, wherein said cell further comprises one or more        deletions or disruptions of a native gene selected from PDC,        ADH, GAL6, CYB2A, CYB2B, GPD, GPP, ALD, and PCK genes.        [B77] The genetically modified yeast cell of paragraph B76,        wherein one or more of the deletions or disruptions results from        insertion of one or more of the exogenous 3-HP pathway genes.        [B78] The genetically modified yeast cell of any of paragraphs        B1-B77, wherein one or more of the exogenous 3-HP pathway genes        are operatively linked to one or more exogenous regulatory        elements.        [B79] The genetically modified yeast cell of paragraph B78,        wherein the one or more regulatory elements are foreign to the        one or more 3-HP pathway genes.        [B80] The genetically modified yeast cell of any of paragraphs        B1-B79, wherein the exogenous PYC gene is operatively linked to        an exogenous promoter that is foreign to the PYC gene.        [B81] The genetically modified yeast cell of any of paragraphs        B1-B80, wherein the exogenous AAT gene is operatively linked to        an exogenous promoter that is foreign to the AAT gene.        [B82] The genetically modified yeast cell of any of paragraphs        B1-B81, wherein the exogenous ADC gene is operatively linked to        an exogenous promoter that is foreign to the ADC gene.        [B83] The genetically modified yeast cell of any of paragraphs        B1-B82, wherein the exogenous BAAT or gabT gene is operatively        linked to an exogenous promoter that is foreign to the BAAT or        gabT gene.        [B84] The genetically modified yeast cell of any of paragraphs        B1-B83, wherein the exogenous 3-HPDH gene is operatively linked        to an exogenous promoter that is foreign to the 3-HPDH gene.        [B85] The genetically modified yeast cell of any of paragraphs        B1-B84, wherein the exogenous PPC gene is operatively linked to        an exogenous promoter that is foreign to the PPC gene.        [B86] The genetically modified yeast cell of any of paragraphs        B1-B85, wherein the cell is capable of growing at a pH of less        than 4 in media containing 75 g/L or greater 3-HP.        [B87] The genetically modified yeast cell of any of paragraphs        B1-B86, wherein the cell is a 3-HP-resistant yeast cell.        [B88] The genetically modified yeast cell of any of paragraphs        B1-B87, wherein the cell has undergone mutation and/or        selection, such that the mutated and/or selected cell possess a        higher degree of resistance to 3-HP than a wild-type cell of the        same species.        [B89] The genetically modified yeast cell of paragraph B88,        wherein the cell has undergone mutation and/or selection before        being genetically modified with the one or more exogenous 3-HP        pathway genes.        [B90] The genetically modified yeast cell of paragraph B88 or        B89, wherein the cell has undergone selection in the presence of        lactic acid or 3-HP.        [B91] The genetically modified yeast cell of paragraph B91,        wherein the selection is chemostat selection.        [B92] A method of producing 3-HP comprising:    -   (i) culturing the genetically modified yeast cell of any of        paragraphs B1-B91 in the presence of medium comprising at least        one carbon source; and    -   (ii) isolating 3-HP from the culture.        [B93] The method of paragraph B92, wherein said carbon source is        selected from glucose, xylose, arabinose, sucrose, fructose,        cellulose, glucose oligomers, and glycerol.        [B94] The method of paragraph B92 or B93, wherein the medium is        at a pH of less than 5, e.g., in the range of about 1.5 to about        4.5, about 2.0 to about 4.0, or about 2.0 to about 3.5.

1. A genetically modified yeast cell comprising an active fermentationpathway that proceeds through intermediates comprisingphosphoenoylpyruvate (PEP) or pyruvate; oxaloacetate (OAA); aspartate;β-alanine; and malonate semialdehyde, wherein the yeast cell furthercomprises an exogenous gene encoding an enzymatically active polypeptidethat catalyzes the conversion of β-alanine to malonate semialdehyde, andwherein the exogenous gene comprises at least 85% sequence identity toan amino acid sequence from SEQ ID NO: 20, 21, or
 24. 2. The geneticallymodified yeast cell of claim 1, wherein the yeast cell is selected froma Crabtree-negative yeast, an Issatchenkia yeast, a Candida yeast, aKluyveromyces yeast, a Pichia yeast, a Schizosaccharomyces yeast, aTorulaspora yeast, a Zygosaccharomyces yeast, or a Saccharomyces yeast.3. The genetically modified yeast cell of claim 2, wherein the yeastcell is selected from the group consisting of Issatchenkia orientalis,Candida lambica, and Saccharomyces bulderi.
 8. The genetically modifiedyeast cell of claim 1, wherein the genetically modified yeast cellfurther comprises an exogenous gene encoding an enzymatically activepolypeptide that catalyzes the conversion of aspartate to β-alanine. 9.The genetically modified yeast cell of claim 8, wherein the exogenousgene encoding the enzymatically active polypeptide that catalyzes theconversion of aspartate to 3-alanine comprises an aspartatedecarboxylase (ADC) gene with at least 50% sequence identity to an aminoacid sequence from SEQ ID NO: 17, 18, 133, 135, 137, or
 139. 10. Thegenetically modified yeast cell of claim 1, wherein the geneticallymodified yeast cell further comprises an exogenous gene encoding anenzymatically active polypeptide that catalyzes the conversion ofoxaloacetate (OAA) to aspartate.
 11. The genetically modified yeast cellof claim 10, wherein the exogenous gene encoding the enzymaticallyactive polypeptide that catalyzes the conversion of oxaloacetate (OAA)to aspartate comprises an aspartate aminotransferase (ATT) gene with atleast 50% sequence identity to an amino acid sequence from SEQ ID NO:14, 15, or
 16. 12. The genetically modified yeast cell of claim 1,wherein the genetically modified yeast cell further comprises anexogenous gene encoding an enzymatically active polypeptide thatcatalyzes the conversion of pyruvate to oxaloacetate (OAA).
 13. Thegenetically modified yeast cell of claim 12, wherein the exogenous geneencoding the enzymatically active polypeptide that catalyzes theconversion of pyruvate to oxaloacetate (OAA) comprises an pyruvatecarboxylase gene (PYK) gene with at least 50% sequence identity to anamino acid sequence from SEQ ID NO: 2, 3, 4, 5, 6, 7, or
 8. 14. Thegenetically modified yeast cell of claim 1, wherein the geneticallymodified yeast cell further comprises an exogenous gene encoding anenzymatically active polypeptide that catalyzes the conversion ofphosphoenoylpyruvate (PEP) to oxaloacetate (OAA).
 15. The geneticallymodified yeast cell of claim 14, wherein the exogenous gene encoding theenzymatically active polypeptide that catalyzes the conversion ofphosphoenoylpyruvate (PEP) to oxaloacetate (OAA) comprises an PEPcarboxylase gene (PCK) gene comprising an amino acid sequence from SEQID NO: 35, 36, 37, 38, or
 39. 16. The genetically modified yeast cell ofclaim 1, wherein the genetically modified yeast cell further comprises adeletion or disruption of one or more native genes involved in ethanolfermentation.
 17. The genetically modified yeast cell of claim 16,wherein the one or more native genes involved in ethanol fermentationcomprises pyruvate decarboxylase (PDC) or alcohol dehydrogenase (ADH).18. The genetically modified yeast cell of claim 1, wherein thegenetically modified yeast cell further comprises a deletion ordisruption of one or more native genes encoding glycerol 3-phosphatedehydrogenase (GPD), glycerol 3-phosphatase (GPP), glycerol kinase,dihydroxyacetone kinase, glycerol dehydrogenase, aldehyde dehydrogenase(ALD), or butanediol dehydrogenase.